Vector-free intracellular delivery by reversible permeabilisation

ABSTRACT

The invention provides a solution to the problem of transfecting non-adherent cells. Devices and delivery compositions containing ethanol and an isotonic salt solution are used for delivery of compounds and compositions to non-adherent cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to U.S.Provisional Patent Application No. 62/438,298 filed Dec. 22, 2016, U.S.Provisional Patent Application No. 62/528,963 filed Jul. 5, 2017, andU.S. Provisional Patent Application No. 62/536,831 filed Jul. 25, 2017,the entire contents of each of which is hereby expressly incorporated byreference herein.

FIELD OF THE INVENTION

The invention relates to the delivery of agents into mammalian cells.

BACKGROUND OF THE INVENTION

Variability in cell transfection efficiency exists among different celltypes. Transfection of suspension cells, e.g., non-adherent cells, hasproven to be very difficult using conventional methods. Thus, a needexists for compositions and methods to facilitate transfection of suchcells.

SUMMARY OF THE INVENTION

The invention provides a solution to the problem of deliveringpayload/cargo compounds and compositions into non-adherent cells.Accordingly, a method of delivering a payload across a plasma membraneof a non-adherent cell comprises the steps of providing a population ofnon-adherent cells and contacting the population of cells with a volumeof an isotonic aqueous solution, the aqueous solution including thepayload and an alcohol at greater than 5 percent (v/v) concentration.For example, the alcohol comprises ethanol, e.g., greater than 10%ethanol. In some examples, the aqueous solution comprises between 20-30%ethanol, e.g., 27% ethanol.

The aqueous solution for delivering cargo to cells comprises a salt,e.g., potassium chloride (KCl) in between 12.5-500 mM. For example, thesolution is isotonic with respect to the cytoplasm of a mammalian cellsuch as a human T cell. Such an exemplary isotonic delivery solution 106mM KCl.

The methods are used to deliver any cargo molecule or molecules tomammalian cells, adherent or non-adherent and are particularly useful todeliver cargo to non-adherent cells because of the difficultiesassociated with doing so prior to the invention. In some examples, thenon-adherent cell comprises a peripheral blood mononuclear cell, e.g.,the non-adherent cell comprises an immune cell such as a T cell (Tlymphocyte). An immune cell such as a T cell is optionally activatedwith a ligand of CD3, CD28, or a combination thereof. For example, theligand is an antibody or antibody fragment that binds to CD3 or CD28 orboth.

The method involves delivering the cargo in the delivery solution to apopulation of non-adherent cells comprising a monolayer. For example,the monolayer is contacted with a spray of aqueous delivery solution.The method delivers the payload/cargo (compound or composition) into thecytoplasm of the cell and wherein the population of cells comprises agreater per cent viability compared to delivery of the payload byelectroporation or nucleofection—a significant advantage of theSoluporation system.

Any compound or composition can be delivered. For example, the payloadcomprises a messenger ribonucleic acid (mRNA), e.g., a mRNA that encodesa gene-editing composition. For example, the gene editing compositionreduces the expression of an immune checkpoint inhibitor such as PD-1 orPD-L1. In some examples, the mRNA encodes a chimeric antigen receptor(CAR).

In certain embodiments, the monolayer of non-adherent/suspension cellsresides on a membrane filter. In some embodiments, the membrane filteris vibrated following contacting the cell monolayer with a spray of thedelivery solution. The membrane filter may be vibrated or agitatedbefore, during, and/or after spraying the cells with the deliverysolution.

Also within the invention is a system comprising: a housing configuredto receive a plate comprising a well; a differential pressure applicatorconfigured to apply a differential pressure to the well; a deliverysolution applicator configured to deliver atomized delivery solution tothe well; a stop solution applicator configured to deliver a stopsolution to the well; and a culture medium applicator configured todeliver a culture medium to the well. A stop solution is one that lacksa cell membrane permeabilizing agent, e.g., ethanol. An examplephosphate buffered saline or any physiologically-compatible buffersolution. The system optionally further comprises: an addressable wellassembly configured to: align the differential pressure applicatoradjacent the well for applying the differential pressure to the well;align the delivery solution applicator adjacent the well for deliveringthe atomized delivery solution to the well; align the stop solutionapplicator adjacent the well to deliver the stop solution to the well;and/or align the culture medium applicator adjacent the well to deliverthe culture medium to the well.

The addressable well assembly can include a movable base-plateconfigured to receive the plate comprising the well and move the platein at least one dimension. The addressable well assembly can include amounting assembly configured to couple to the delivery solutionapplicator, the stop solution applicator and the culture mediumapplicator.

The delivery solution applicator can include a nebulizer. The deliverysolution applicator can be configured to deliver 10-300 micro liters ofthe delivery solution per actuation.

The system can include a temperature control system configured tocontrol a temperature of the delivery solution and/or of the platecomprising the well.

The system can include an enclosure configured to control an environmentof the plate comprising the well.

The differential pressure applicator can include a nozzle assemblyconfigured to form a seal with an opening of the well and to deliver avapor to the well to increase or decrease pressure within the well,thereby driving a liquid portion of the culture medium from the wellsuch that a layer of cells remains within the well.

The stop solution applicator can comprise a needle emitter configured tocouple to a stop solution reservoir.

The culture medium applicator can comprise a needle emitter configuredto couple to a culture medium reservoir.

The system can further comprise a controller configured to: receive userinput; operate the delivery solution applicator to deliver the atomizeddelivery solution to a cellular monolayer within the well; incubate, fora first incubation period, the cellular monolayer after application ofthe delivery solution; operate, in response to expiration of the firstincubation period, the stop solution applicator to deliver the stopsolution to the cellular monolayer; and incubate, for a secondincubation period and in response to application of the stop solution,the cellular monolayer. The controller can be further configured to:iterate operation of the delivery solution applicator, incubation forthe first incubation period, operation of the stop solution applicator,and incubation for the second incubation period for a predeterminednumber of iterations.

The system can further comprise a controller configured to: operate thepositive pressure system to remove supernatant from the well to create acellular monolayer within the well.

The delivery solution applicator can include a spray head and a collarencircling a distal end of the spray head, wherein the collar isconfigured to prevent contamination between wells in a multi-well plate,wherein the collar is configured to provide a gap between the plate andthe collar.

The delivery solution applicator can include a spray head and a filmencircling a distal end of the spray head.

The system can further comprise a vibration system coupled to a membraneholder and configured to vibrate a membrane.

The system can further comprise the plate, wherein the well isconfigured to contain a population of non-adherent cells.

The delivery solution includes an isotonic aqueous solution, the aqueoussolution including the payload and an alcohol at greater than 5 percent(v/v) concentration. The alcohol can comprise ethanol. The aqueoussolution can comprise greater than 10% ethanol. The aqueous solution cancomprise between 20-30% ethanol. The aqueous solution can comprise 27%ethanol. The aqueous solution can comprise between 12.5-500 mM KCl. Theaqueous solution can comprise between 106 mM KCl.

The non-adherent cells can comprise a peripheral blood mononuclear cell.The non-adherent cells can comprise an immune cell. The non-adherentcells can comprise non-adherent cell comprises a T lymphocyte. Thepopulation of non-adherent cells can comprise a monolayer.

The payload can comprise a messenger ribonucleic acid (mRNA). The mRNAcan encode a gene-editing composition. For example, the gene editingcomposition reduces the expression of PD-1. The mRNA can encode achimeric antigen receptor.

The system can be used to deliver a cargo compound or composition to amammalian cell.

In another aspect, a composition comprises an isotonic aqueous solution,the aqueous solution comprising KCl at a concentration of 10-500 mM andethanol at greater than 5 percent (v/v) concentration for use to delivera cargo compound or composition to a mammalian cell. The KClconcentration can be 106 mM and said alcohol concentration can be 27%.

The compounds that are loaded into the MPS composition are processed orpurified. For example, polynucleotides, polypeptides, or other agentsare purified and/or isolated. Specifically, as used herein, an“isolated” or “purified” nucleic acid molecule, polynucleotide,polypeptide, or protein, is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.Purified compounds are at least 60% by weight (dry weight) the compoundof interest. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight thecompound of interest. For example, a purified compound is one that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of thedesired compound by weight. Purity is measured by any appropriatestandard method, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA)or deoxyribonucleic acid (DNA)) is free of the genes or sequences thatflank it in its naturally-occurring state. Purified also defines adegree of sterility that is safe for administration to a human subject,e.g., lacking infectious or toxic agents. In the case of tumor antigens,the antigen may be purified or a processed preparation such as a tumorcell lysate.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

A small molecule is a compound that is less than 2000 Daltons in mass.The molecular mass of the small molecule is preferably less than 1000Daltons, more preferably less than 600 Daltons, e.g., the compound isless than 500 Daltons, 400 Daltons, 300 Daltons, 200 Daltons, or 100Daltons.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Genbank and NCBI submissions indicated by accessionnumber cited herein are incorporated herein by reference. All otherpublished references, documents, manuscripts and scientific literaturecited herein are incorporated herein by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph illustrating the assessment of feeder cells as amethod of T cell activation. GFP mRNA was delivered to T cells culturedin cRPMI medium.

FIG. 2A is a bar graph illustrating results of RPMI media comparingStemCellTech reagent vs Dynabeads at 2 concentrations.

FIG. 2B is a bar graph illustrating that the experiment (from FIG. 2A)was repeated to include StemCell tech reagent at 3 times recommendedconcentration but didn't have any effect.

FIG. 3 is a bar graph illustrating the improvement in uptake using PrimeXV culture medium and activation with Dynabeads (3:1 bead to cell).

FIG. 4A is a bar graph illustrating poor cell recovery when cultured inPrime XV medium.

FIG. 4B are images depicting poor cell recovery and variability in cellproliferation when cultured in Prime XV. Cells were activated usingDynabeads (3:1).

FIG. 5 is a bar graph illustrating results of Dynabead-activated T cellscultured in Immunocult culture media.

FIG. 6 is a bar graph illustrating the optimum post-activation windowfor delivery of mRNA to T cells using Dynabeads as activation reagent.

FIG. 7 is a bar graph illustrating TransAct vs Dynabead in Immunoculttime course.

FIG. 8 is a bar graph illustrating results of TexMACS vs Immunocultusing TransAct as T cell activator.

FIG. 9 is a bar graph illustrating results of cells left to recoverprior to activation demonstrated higher uptake efficiency to thoseactivated immediately post-thaw.

FIG. 10 is a bar graph illustrating results of cells cultured at ahigher cell density prior to delivery using the vector-free deliverytechnology. A higher cell density (5×10⁶ /ml) correlated with higheruptake efficiency.

FIG. 11 is a bar graph illustrating that the addition of zinc chlorideto the culture media resulted in higher uptake efficiency.

FIG. 12 is a bar graph showing that multiple hits on CD4+ T cells werenegatively selected using CD8 microbeads. Cells were transfected withGFP mRNA by soluporation. Multiple hits improved uptake over 3 donorstested.

FIG. 13 is a bar graph showing expression of transfected mRNA byPBMC-initiated T cells. T cells were enriched from PBMC for 2-3 days.Cells were transfected with GFP mRNA by soluporation.

FIG. 14A is a diagram, FIG. 14B is a photograph, and FIG. 14C is aphotograph showing membrane inserts. An example image of a ThinCert12-well insert (FIG. 14A). An image of the insert device whereby theinsert would be placed within the O-ring as indicated by the arrow (FIG.14B). The device attached to a syringe to allow a vacuum to be appliedto the insert to remove the media (FIG. 14C).

FIG. 15 is a bar graph showing Solupore delivery of cargo to suspensioncells (non-adherent cells). Fluorescently-labelled beta-lactoglobulin(BLG), bovine serum albumin (BSA) or ovalbumin (OVA) was delivered to amonolayer of Jurkat cells formed using the insert device. Expressionlevels were analysed by flow cytometry.

FIG. 16A is a photograph of microtiters plates (PES filter plate), FIG.16B and FIG. 16C are dot plots showing expression of product fromtransfected mRNA. An example image of the Acroprep Advance, Supormembrane, 96-well filter plate (FIG. 16A). GFP expression levels from 5random donors across 5 experiments 24 hr post mRNA delivery to T cells(FIG. 16B). Relative viability of T cells 24 hr post mRNA delivery to Tcells (FIG. 16C).

FIG. 17 is a bar graph showing the results of cargo delivery using aPCTE filter plate. Viability and GFP expression 24 hr post mRNA deliveryto T cells using a PCTE 96-well plate.

FIG. 18 is bar graph showing mRNA expression after cargo delivery tocells (comparison of 96-well filter plates). GFP expression in T cellsfollowing mRNA delivery. A monolayer of cells were formed in either aPCTE or PES filter plate.

FIG. 19A-FIG. 19B are bar graphs showing expression of transfected mRNA.A comparison of media removal methods. Media was removed from cellsseeded in a 96-well filter plate by either centrifugation or vacuumpressure. (FIG. 19A). Media was removed from cells seeded in a 96-wellfilter plate by either centrifugation or positive pressure (FIG. 19B).GFP mRNA was delivered by Soluporation and expression assessed by flowcytometry.

FIG. 20A and FIG. 20B are photographs of cells. Monolayer formationusing Dynabead bound cells (FIG. 20A). Media was removed by pipettingand GFP mRNA was delivered by Soluporation. GFP expression was detected24 hr later by fluorescence microscopy (FIG. 20B).

FIG. 21A is a series of photographic images illustrating the delivery ofGFP mRNA to MSCs. GFP RNA was delivered to BM-MSCs and iPSC-MSCs usingthe vector-free reversible permeabilization method and analysed byfluorescence microscopy (10× magnification).

FIG. 21B is a bar graph illustrating delivery of GFP mRNA by MSCsanalysed by flow cytometry; n=3, data are depicted as the mean±standarddeviation.

FIG. 22A is a table and FIG. 22B is a photograph of cells showing agreen fluorescent marker. Uptake of Alexa 488 Dextran 10KD using theultrasonic nebulizer. Representative data demonstrating uptake ofdextran into U2OS cells using the ultrasonic nebulizer (a) Tablesummarising the data where up to 64% dextran uptake was achieved and (b)Fluorescence micrograph showing green Dextran-Alex488.

FIG. 23 is a photograph of an Ari Mist Nebulizer payload fluidic controlusing a pinch valve. Image demonstrates the fluidic control of thepayload solution to the Ari Mist nebulizer on the test rig used foroptimisation of atomisation of the delivery solution. The ari mistnebulizer (1) is connected to a pinch valve (2) connected to an Elveflowdelivery solution reservoir (3).

FIG. 24 is a dot plot showing Ari Mist Test Rig Longitudinal Data. Graphdemonstrates GFP mRNA efficiency to T-cells using the Ari Mist nebulizergathered over the course of the optimisation experiments. The datademonstrates average uptake efficiency of up to 25% and highreproducibility across replicates.

FIG. 25 is a dot plot showing Ultrasonic 180 kHz Test Rig LongitudinalData. Graph demonstrates GFP mRNA efficiency to T-cells using theUltrasonic nebulizer gathered over the course of the optimisationexperiments. The data demonstrates average uptake efficiency of up to30% and high reproducibility across replicate.

FIG. 26 is a dot plot showing Nasal Head Test Rig Longitudinal Data.Graph demonstrates GFP mRNA efficiency to T-cells using the MAD nasalhead nebulizer gathered over the course of the optimisation experiments.The data demonstrates average uptake efficiency of up to 22% withreduced reproducibility across replicates compared to the Ari Mist andUltrasonic spray head.

FIG. 27A and FIG. 27B are bar graphs showing results from a comparisonof atomiser height above cells. GFP expression was assessed in T cellsfollowing delivery of mRNA with the atomiser height set at 31, 26, 12 or11 mm.

FIG. 28 is a bar graph showing a comparison of delivery solutionvolumes. GFP expression in T cells following delivery of GFP mRNA indelivery solution of different volumes (4, 1 or 0.5 μl).

FIG. 29 is a bar graph showing a comparison of salt concentrations. GFPmRNA was added to delivery solution containing 12 mM KCl (Hypotonic) or106 mM KCl (Isotonic). “Hypotonic” and “isotonic” refer to tonicityrelative to cell cytoplasm. Expressions levels at 24 hr were assessed byflow cytometry.

FIG. 30 is a bar graph showing a comparison of the number of “Hits”. Tcells were sprayed (Hit) once, twice or three times, with a 2 hourincubation in between each spray. GFP expression was assessed by flowcytometry 24 hr later.

FIG. 31 is a bar graph showing a comparison of T cell seeding densities.T cells were seeded at 1.25, 2.5, 3.5, 5 and 7.5×10⁵ cells per well andGFP mRNA delivered by Soluporation. GFP expression was assessed at 24 hrby flow cytometry.

FIG. 32 is a line graphs showing distribution of cell diameter. AverageT cell diameter (9.5 μm) at 24 hr post addition of DynaBeads (3:1 Beadto cell ratio).

FIG. 33A-FIG. 33C are bar graphs showing delivery and viability ofAvectas (Soluporation) compared with electroporation. At (FIG. 33A)delivery efficiency of 10 kDa dextran-Alexa488 to A549 cells was 52.8%(+/−2.7%) as quantified by flow cytometry and cell survival comparedwith untreated control cells was 78.3% (+/−4.1%) as determined bypropidium iodide exclusion and flow cytometry. (FIG. 33B) Forelectroporation, delivery efficiency was 92.9% (+/−0.6%) and cellsurvival was 73.0% (+/−9.8%). (FIG. 33C) The transfection score((transfected cells/total cells)×(viable cells/total cells)) was 0.33(+/−0.05) for the current subject matter technology and 0.51 (+/−0.13)for electroporation with no significant difference between the scores,p=0.25.

FIG. 34 is a bar graph showing a comparison of Soluporation andNucleofection. Human T cells were transfected with GFP mRNA using eitherSolupore or Nucleofection. GFP expression and cell viability (7-AAD) wasanalysed 24 hr later by flow cytometry.

FIG. 35A is a bar graph, FIG. 35B is a line graph, and FIGS. 35C and 35Dare photographs showing a comparison of fluorescence intensity betweenSolupore and Nucleofection-mediated delivery of cargo. The Medianfluorescence intensity produced by human T cells 24 hr post-delivery ofGFP mRNA was compared between Solupore and Nucleofection (FIG. 35A) anda histogram showing the increase in intensity (FIG. 35B). An exampleimage of T cells aggregates expressing GFP following Soluporation(brighter) and Nucleofection.

FIG. 36 is a dot plot showing a comparison of fluorescence intensity andGFP expression between Solupore and Nucleofection with different amountsof mRNA delivered. A dose response in the Median fluorescence intensityproduced by human T cells 24 hr post delivery of different amounts ofGFP mRNA was observed post Soluporation and Nucleofection.

FIG. 37A is a photograph, and FIG. 37B, 37C, and FIG. 37D are bar graphsshowing evaluation of delivery of cargo into cells. Diffusion of cargointo cells and resealing of plasma membrane. (FIG. 37A) 10 kDadextran-Alexa488 was delivered into A549 cells and analysed byfluorescence microscopy at 30 sec, 1 min, 2 min and 3 min post-delivery(10× mag.). (FIG. 37B) A549 were pretreated with Dynasore (4 mM) orchloropromazine (20 μM) to inhibit clathrin-mediated endocytosis orNystatin (20 μg/ml) or EIPA (100 μM) to inhibit caveolar-mediatedendocytosis and micropinocytosis respectively (n=3). None of theseinhibitors blocked the uptake of EGFP mRNA. (FIG. 37C) Lipofectamine2000 was used as a positive control for endocytosis-mediated delivery.GFP expression was significantly reduced in lipofected cells treatedwith Dynasore (n=3). (FIG. 37D) To examine recovery of the cell membraneafter permeabilization, delivery solution was sprayed onto A549 cells inthe absence of cargo and at subsequent time points (0 to 182.5 min)medium was removed and 50 μl propidium iodide (100 μg/ml) in PBS added.After 2 min incubation the PI solution was removed and the cells wereharvested. For basal levels of PI uptake, untreated cells received 50 μlPI in PBS. PI uptake was analysed by flow cytometry and the dataindicate that the cells remain permeable to PI for up to 6 minpost-treatment but then reseal and prevent uptake thereafter (n=3).Error bars represent standard error (SE) across three experiments (n=3).***p<0.001, student's t test for independent means.

FIG. 38A is a bar graph illustrating the PD-1 surface expression on Tcells. Data shown are the mean of five independent experiments wherecontrol group is normalised to 100%.

FIG. 38B are photographic images illustrating PD-1 surface expression onT cells determined at 72 h post-transfection by flow cytometry. Datashown is from one independent experiment.

FIG. 39 are photographic images showing that T cell clones at day 4 posttreatment (see arrows) by light microscope, indicating goodproliferation and activation of the T cells in untreated controls andthe vector-free intracellular delivery method described herein treatedcells. Significantly fewer clones were observed in those cells thatunderwent electroporation. Data shown is one independent experiment.

FIG. 40 is a schematic of CAR construction of Lenti-T7-CD19-3rd-CARvector.

FIG. 41A is a schematic depicting the sequence of single-chain variablefragment (scFv).

FIG. 41B is a schematic depicting the nucleotide sequence (codonoptimized) of the CAR cassette.

FIG. 41C is a schematic depicting the protein sequence of the CARcassette.

FIG. 42 is a photographic image showing the Restriction Digestion Map ofthe CAR vector.

FIG. 43 is a table showing the QC results and Certificate of Analysis ofthe commercially sourced CAR vector.

FIG. 44A-FIG. 44B are is a schematic depicting the CAR sequencealignment validation of the commercially sourced CAR vector.

FIG. 45 is a dot plot of flow cytometry data showing CAR expression inhuman primary T cells following mRNA delivery by the vector-freedelivery technology described herein.

FIG. 46A is a photograph of an electrophoretic gel and FIG. 46B is a bargraph showing supercoiled vs open circular nucleic acids.

FIG. 47A-FIG. 47B are dot plots showing the effect of transfectionmethods on expression of CD4 and CD8 on T cells. The expression of cellsurface CD4 and CD8 was examined 6 hr and 24 hr after eithernucleofection, electroporation (Neon) or Soluporation. At 6 hr,expression levels were similar to untreated control cells. However, at24 hr, expression in soluporated cells and nucleofected cells wassimilar to control untreated cells whereas expression was significantlyreduced in electroporated cells.

FIG. 48A-FIG. 48C are dot plots showing a mRNA microarray analysis.Cells were transfected with GFP mRNA. The highest level of geneexpression changes occurred in Neon electroporation treatments. Of the20,893 genes analysed, Solupore had 32 changed, nucleofection had 24changed and electroporation had 317 changed.

FIG. 49 is a line graph showing cell proliferation. T cells weretransfected with GFP mRNA and cells were counted each subsequent day for7 days. Proliferation rates in soluporated and nucleofected cells weresimilar to untreated control cells whereas the ability of Neonelectroporated cells was reduced compared with control cells.

FIG. 50 is a line graph showing cell proliferationpost-cryopreservation. T cells were transfected with GFP mRNA and cellswere cryopreserved in 10% DMSO and foetal bovine serum 24 hrspost-transfection. Cells were thawed and seeded at 0.5×10⁶/ml on day 0in Immunocult media+IL-2. Cells were counted and re-seeded by addingadditional media each day for 5 days. Proliferation rates in soluporatedand nucleofected cells were similar to untreated control cells whereasthe ability of electroporated (e.g., Neon) cells was reduced comparedwith control cells.

FIG. 51 is a series of bar graphs showing Interferon-gamma (IFNg)production. IFNg production, measured at 14 days post transfection, wasnot reduced in T cells following soluporation, nucleofection orelectroporation compared with control cells.

FIG. 52 is a bar graph showing IFNg production. Soluporated,nucleofected, electroporated and untreated cells were cryopreserved 24 hpost-transfection. Cells were then re-stimulated with either Dynabeadsor a PMA/Ionomycin cocktail for 4 hrs and their supernatants wereanalysis for IFN-γ production.

FIG. 53A-FIG. 53C are bar graphs showing evaluation of CD 45⁺ cellspost-treatment. Analysis of blood at Day 14 following injection of humanPBMC into NSG mice. FIG. 53A. Human CD45+ cells were detected in micethat received untreated (UT) PBMC and soluporated (Sol) PBMC. Lownumbers of human CD45+ cells were detected in mice that receivednucleofected (Nuc) PBMC. FIG. 53B. The presence of CD4+ cells wasconfirmed in Groups 2, 5 and 6. FIG. 53C. The presence of CD8+ cells wasconfirmed in Groups 2, 5 and 6.

FIG. 54 is a line graph showing calibration data for the deliverysolution using the elveflow-pinch valve system. Graph demonstratescalibration data for repeated atomisation of 4 μl volume of deliverysolution. The orange bar shows the volume measured from 11 repeat spraysof delivery solution where the expected volume was 4 μl. Datademonstrates a relative standard deviation (% RSD) of 9.38% at thisvolume range. The pink bar shows the volume measured from 9 repeatsprays following re-load of the sample reservoir. These data highlightthe limitations with the elveflow-pinch valve system which include lackof precision and accuracy when spraying 4 μl volumes and a lack ofcalibration holding following re-load of the system.

FIG. 55 is a is a line graph showing Force Sensor Parametrisation 1. Aforce sensor was placed at 31, 26 or 11 mm below the Ari Mist atomiserand the pressure of air driven through the atomiser adjusted from 0.5Bar to 2 Bar. The amount of liquid dispensed was also altered byadjusting the amount of pressure applied to the Elveflow system. Thepeak pressure experienced by the sensor is measured in Volts.

FIG. 56 is a series of line graphs showing Force Sensor Parametrisation2. A force sensor was placed at 31, 26 or 11 mm below the Arimistatomiser and the pressure of air driven through the atomiser adjustedfrom 1.15 Bar to 2.15 Bar. The peak pressure experienced by the sensoris measured in Volts.

FIG. 57 is a bar graph showing Force Sensor GFP Delivery. GFPexpression, Viability (Live) and Peak Pressure (Volt) depicted followingGFP mRNA delivery with variable air pressures and volumes.

FIG. 58 is a photograph of a Certus Flex set-up. Image demonstratesCertus flex set-up with delivery solution loaded in Channel 1, Stopsolution loaded in Channel 2 and Culture medium loaded in Channel 3.

FIG. 59 is a diagram of a Droplet Array Pattern. Figure demonstrates thedroplet array pattern tested and is further described in Table 1. Thetotal volume delivered into the well was 2 μl or 7 μl and the number ofdroplets increased from 4 to 25. This corresponded to droplet volumesranging from 0.08 to 0.5 μl.

FIG. 60A is a diagram and FIG. 60B is a bar graph a seeding mask and theeffect of a seeding mask, respectively. A comparison of GFP mRNAexpression in cells that were seeded into a well of a PCTE plate in thepresence or absence of a seeding mask (FIG. 60A). GFP expression wasmeasured 24 hr post delivery (FIG. 60B).

FIG. 61A is a bar graph and FIG. 61B is a table showing datademonstrating lack of GFP mRNA delivery using the Certus Flex DigitalDispensing technology. (FIG. 61A) Graph depicts representative datademonstrating lack of mRNA delivery using the Certus Flex. Cellviability was not adversely affected using this system. (FIG. 61B)Corresponding data set detailed in table.

FIG. 62A is a bar graph showing the effect of a collar and FIG. 62B is aphotograph showing a collar. Testing of an Enclosing collar on the AriMist spray head. (FIG. 62A). Graph demonstrates data comparing deliveryefficiency without an enclosing collar (− collar), with the enclosingcollar (+ collar) and with the enclosing collar with a 1 mm gap betweenthe collar and the 96-well plate (+ collar and 1 mm gap). Resultsindicate the addition of the enclosing collar had a negative impact onthe spray which was reversed when a 1 mm gap was left between the collarand the well plate. (FIG. 62B) Image demonstrates the enclosing collarset-up. The collar is inserted onto the Ari Mist spray head. The sprayhead is positioned 27 mm over the well of a double height plate whichleaves a 1 mm gap between the collar and the top of the well plate.Delivery efficiency was not impacted with this set-up.

FIG. 63A is a bar graph showing the results of testing with a Gygervalve, and FIG. 63B is a photographic image of X-pierce film. Graphpresents data from three independent experiments comparing deliveryefficiency on Test Rig 1 (which utilises a Clippard pinch valve), TestRig 3 (which utilizes a Gyger micro valve) and Test rig 3 and anX-pierce film on a PCTE filter plate. Note: only two experiments weredone comparing the TR3 and Gyger valve only (TR3 Gyger). The resultsdemonstrate increased delivery efficiency when the Gyger micro valve(TR3 Gyger) was used in place of the Clippard valve (TR1). The additionof the X-pierce film on the PCTE plate did not have any effect on thisdelivery efficiency (TR3 Gyger+enclosing). Each bar represents a singleexperiment with a minimum of 4 replicates.

FIG. 64 is a process flow diagram illustrating an example processaccording to some aspects of the current subject matter.

FIG. 65 is a diagram illustrating one embodiment of a delivery system

FIG. 66 is a diagram that illustrates 9 elements of a delivery system.

FIG. 67 is a side view of an embodiment of a delivery system.

FIG. 68 is perspective of a portion of an embodiment of a deliverysystem.

FIG. 69 is an enlarged perspective view of the delivery system shown inFIG. 68.

FIG. 70 is an exploded top perspective view of a vacuum manifoldassembly of the delivery system shown in FIG. 68.

FIG. 71 is an exploded bottom perspective view of a vacuum manifoldassembly of the delivery system shown in FIG. 68.

FIG. 72 show a top view of a base plate of the vacuum manifold assemblyshown in FIG. 70.

FIG. 73 is a side cross-sectional view of the base plate shown in FIG.72.

FIG. 74 is a bottom view of a top plate of the vacuum manifold assemblyshown in FIG. 72.

FIG. 75 is a side cross-sectional view of the top plate shown in FIG.74.

FIG. 76 is a top view of the top plate shown in FIG. 74.

FIG. 77 is a top view of a well filter plate of the vacuum manifoldassembly shown in FIG. 72.

FIG. 78 is another top view of the well filter plate shown in FIG. 72,where the well filter plate has been rotated 180°.

FIG. 79 is a front perspective view of a portion of the precision rigsystem shown in FIG. 67.

FIG. 80 is a back perspective view of a portion of the precision rigsystem shown in FIG. 67.

FIG. 81 is a top view of a portion of the precision rig system shown inFIG. 67.

FIG. 82 is a distribution of GlowGerm particles that were observedfollowing centrifugation and vacuum extraction.

FIG. 83 is a diagram that illustrates six elements of a positivepressure delivery system.

FIG. 84 is a perspective view of an embodiment of positive pressuredelivery system that includes a manifold assembly, a mounting arrayhaving modular fluidic head modules, an X-Y actuator, and a controlsystem.

FIG. 85 is an enlarged view of a portion of the positive pressuredelivery system shown in FIG. 84.

FIG. 86 is an enlarged view of a modular fluidic head module thatenables movement in a vertical direction.

FIG. 87 is the modular fluidic head module shown in FIG. 86, with aneedle assembly attached thereto. The needle assembly is used todispense a culture medium and a stop solution.

FIG. 88 is the modular fluidic head module shown in FIG. 86, with anebulizer assembly attached thereto. The nebulizer assembly is used foratomization of a payload and delivery solution to deliver the solutionto cells.

FIG. 89 modular fluidic head module shown in FIG. 86, with a positivepressure nozzle assembly attached thereto. The positive pressure nozzleassembly is used for removal of a culture medium.

FIG. 90 is a side view of a positive pressure nozzle of the positivepressure system shown in FIGS. 84-85, used for removal of a culturemedium. FIG. 90 illustrates a distal end of the nozzle as it contacts awell of a 96-well filter plate.

FIG. 91 is an enlarged view of a portion of the positive pressure nozzleshown in FIG. 90. The positive pressure nozzle is shown to form a sealwith the well.

FIG. 92 is an exemplary embodiment of a portion of a nozzle assemblythat includes a valve to control delivery of air to a well of a filterplate.

FIG. 93 is an embodiment of a nebulizer assembly used for optimizationof atomization of a delivery solution. The nebulizer assembly includes asyringe, a micro valve, and a nebulizer. The nebulizer is be coupled tothe micro valve via a coupling element (e.g., a precolumn coupler). Themicro valve is retained within, and/or coupled to, a valve holder, whichis be coupled to the syringe via an adapter. The nebulizer assemblyenables high accuracy and precision of delivery of payload solutions tothe nebulizer.

FIG. 94 is a plot showing efficiencies of payload delivery for andultrasonic emitter operating at 180 kHz.

FIG. 95 is a plot showing a series of data characterizing GFP uptake foran ultrasonic emitter, an Ari Mist nebulizer, and a MAD nasal sprayemitter. The data demonstrates GFP delivery efficiency of 32.7%, 24.8%and 16.9% with the ultrasonic emitter (180 Hz), Ari Mist nebulizer, andthe MAD nasal spray emitter, respectively.

FIG. 96 is a plot showing a series of data characterizing cell viabilityfor the ultrasonic emitter, the Ari Mist nebulizer, and the MAD nasalspray emitter. The data demonstrates relative cell viability of 72.3%,85.3% and 99.7% with the ultrasonic (180 Hz) nebulizer, Ari Mistnebulizer and the MAD nasal nebulizer, respectively. The data representa minimum of 3 technical repeats for each spray head tested.

FIG. 97 is an exemplary embodiment of a nebulizer assembly that includesan enclosing collar positioned around a spray head of an Ari Mistnebulizer. The collar is inserted onto the Ari Mist Spray head, and thespray head is positioned 27 mm from the base of a well of a filterplate, thereby leaving a 1 mm gap between the collar and an uppersurface of the filter plate.

FIG. 98 is a plot showing data characterizing efficiency (GFP uptake)corresponding to a spray head without a collar, a spray head with acollar that forms a seal with a filter plate, and a spray head with thecollar where a 1 mm gap exists between the collar and the filter plate.The data indicate that use of the collar that formed a seal with thefilter plate had a negative impact on the spray. The collar that waspositioned 1 mm above the filter plate did not impact the spray.

FIG. 99 is an exemplary embodiment of a 96-well PCTE filter plate withan X-pierce film adhered to an upper surface of the filter plate.

FIG. 100 is a plot showing data characterizing efficiency (GFP uptake)corresponding to tests performed with rig 1 (R1) with unenclosed filterplate, tests performed with rig 3 (R3) with an unenclosed filter plate,and tests performed with R3 with an filter plate that included X-piercefilm enclosure over the wells of the filter plate. R1 utilizes aclippard pinch valve to control flow, and R3 utilizes a Gyger microvalve. The data indicates increased delivery efficiency when the Gygermicro valve (R3 Gyger) was used in place of the Clippard valve (R1). Theaddition of an X-Pierce film on the PCTE filter plate did not have anyeffect on the delivery efficiency. Each bar represents a singleexperiment with a minimum of 4 replicates.

FIG. 101 is an embodiment of a heating system that can be used heat adelivery solution, stop solution, and culture medium;

FIG. 102 is a side view of an embodiment of a cooling system that can beused cool the delivery solution, stop solution, and culture medium.

FIG. 103 is a perspective view of an embodiment of a cooling system thatcan be used cool the delivery solution, stop solution, and culturemedium.

FIG. 104 is a perspective view of an embodiment of a mounting assemblythat can releas ably retain needle emitters and an ultrasonic atomizer

FIG. 105 is an exploded perspective view of a portion of the mountingassembly shown in FIG. 90.

FIG. 106 is a perspective view of another embodiment of a mountingassembly that can releas ably retain needle emitters and an ultrasonicatomizer

FIG. 107 is a perspective view of an embodiment of a mounting assemblythat can releas ably retain needle emitters and a nebulizer.

FIG. 108 is an exploded view of an exemplary embodiment of a stirredcell system configured to facilitate forming a monolayer of cells. Thestirred cell system is assembled by inserting a membrane into a membraneholder, inserting the membrane holder into a base, and screwing a bodyof the system into the base. Cells (e.g., a culture medium containingcells) are delivered to a chamber formed by the base and the body, and acap is screwed is screwed onto the body opposite the base to enclose thechamber. Positive pressure in the range of 50-100 mbar is delivered tothe chamber via a pressure inlet tubing coupled to the cap. The pressureis applied for 10-60 seconds.

FIG. 109 is a top view the membrane holder of the stirred cell systemshown in FIG. 108.

FIG. 110 is a top view of another embodiment of a membrane holder thatincludes holes, but no ridges.

FIG. 111 is a top view of a membrane that was used with the membraneholder shown in FIG. 110 during tests to assess an impact of the designof the membrane holder on formation of a cell monolayer. Dynabeads wereused in place of cells. The results indicate that the Dynabeads pooledat locations of the membrane corresponding to locations of holes in themembrane holder.

FIG. 112 is a top view of another embodiment of a membrane holder thatincludes holes, concentric channels, and straight channels.

FIG. 113 is a top view of a membrane that was used with the membraneholder shown in FIG. 112 during tests to assess an impact of the designof the membrane holder on formation of a cell monolayer. Dynabeads wereused in place of cells. The results indicate that the membrane holdergenerates an even distribution of Dynabeads.

FIG. 114 is a perspective view of another embodiment of a membraneholder configured to facilitate effective removal of a culture mediumand formation of a cell monolayer.

FIG. 115 is an exemplary embodiment of a nebulizer assembly of asolution delivery system, also referred to as rig 4 (R4). The nebulizerassembly includes a nebulizer (e.g., a LB-100 spray head), a couplingelement (e.g., an IDEX connection) configured to facilitate deliveringair and liquid (e.g., the permeabilizing solution) to the nebulizer, asolution reservoir (e.g., an Elveflow sample reservoir) configured toprovide the permeabilizing solution to the nebulizer, and a pinch valveconfigured to control delivery of the permeabilizing solution to thenebulizer.

FIG. 116 is a perspective view of a mounting system of a solutiondelivery system. The nebulizer assembly shown in FIG. 115 can be mountedto the mounting system. The mounting system can include a valve andreservoir mount, a spray head mount, and a nebulizer retaining collar toaccommodate the nebulizer.

FIG. 117 is a perspective view of a solution delivery systemillustrating the nebulizer assembly shown in FIG. 115 mounted to themounting system shown in FIG. 116.

FIG. 118 is a side view of a solution delivery system illustrating thenebulizer assembly shown in FIG. 115 mounted to the mounting systemshown in FIG. 116.

FIG. 119 is a side view of an exemplary closed stirred cell system.Shown are three component parts a) chamber lid, b) chamber wall and c)chamber base. Part a) the lid, is made from 316 stainless steel andaccommodates the LB-100 spray head and inlets for addition of cellsuspension, stop solution and culture medium. Part b) the wall, ismanufactured from glass and includes inlets which can allow forintroduction of a cell suspension through a cell introducer and includesports which can permit entry of instrumentation to enable monitoring ofhumidity or gases. The wall is designed to allow for expansion of airthrough inclusion of an expansion port, which allows addition of anexpansion chamber or through inclusion of expansion space by designingin an area of the wall with a wider diameter. Part c) the base, ismanufactured from 316 stainless steel and will accommodate the custommembrane holder. To connect Part a to b and b to c are mating NW-KFflanges secured with a stainless steel clasp compliant with GMPmanufacturing.

FIG. 120 is a plot showing data that characterizes force profiles for aLB-100 nebulizer corresponding to various test parameters.

FIG. 121 is a plot showing data characterizing cell recovery andviability following cell monolayer formation. Representative datademonstrates cell recovery from PES and PCTE filter membranes. A cellsuspension at two different concentrations was applied to a 63 mmstirred cell unit. Pressure in the range of 100-250 mbar was applied for10 seconds. Following formation of the cell monolayer, the filter wasrinsed and cells counted and viability analysed. Data shows cellrecovery and viability is improved from the PCTE (track-edged) filters.

FIG. 122 is another plot showing data characterizing cell recover andviability following cell monolayer formation.

FIG. 123 is another plot showing data characterizing deliver efficiencyusing a LB-100 nebulizer. Representative data demonstrates deliveryefficiency of GFP mRNA into T-cells using the LB-100 nebulizer acrossdifferent parameters tested (100 mm distance, 420 ms spray duration, 600mbar pressure corresponding to 100 μl volume delivered and 2.5 bar airpressure). In addition, the presence of walls and an enclosing film wastested. 100 μl delivery solution was atomised using the LB-100 onto amonolayer of 20×10⁶ T-cells in a 40 mm diameter target area. Deliveryefficiency of up to 50% was achieved with a single spray and up to 58%with a double spray protocol. Average viability across treatment groupswas 60%.

FIG. 124 a diagram illustrating a mechanism of action.

FIG. 125 is an embodiment of an Ari Mist nebulizer. Shown here are theconnections for liquid and air to the nebulizer. Described in U.S. Pat.Nos. 5,411,208; 6,634,572 and Canadian Patents # 2,112,093 & 2,384,201.

FIG. 126 is an exemplary embodiment of a payload delivery system (Rig1).

FIG. 127 is an exemplary embodiment of a payload delivery system (Rig3).

FIG. 128 is an exemplary embodiment of a payload delivery system (Rig4).

FIG. 129 is a schematic of a software platform including graphical userinterface used to output a program to a PLC which provides local controlto the mechanical and electrical system.

FIG. 130 is example of graphical user interface experiment canvasaccording to some aspects of the current subject matter. The userinterface includes an experiment canvas which allows the user to varyparameters. The parameters which can be varied by the user include thelocation and number of wells to be addressed, the sequence of stepsincluding vacuum or positive pressure, dispense of payload, stopsolution and culture medium and the volume delivered. The user can alsomodify the actuator speed and the incubation times.

FIG. 131 is exemplary embodiment of a closed stirred cell systemconfigured to facilitate forming a monolayer of cells. As shown in theillustrated example, the closed stirred cell system 12200 includes a cap12202 a body 12204 which forms a chamber wall, a cell introducer 12203,and a chamber base 12208, which includes a membrane holder.

FIG. 132 is an exemplary Midi system. Shown is the 63 mm stirred cellunit containing the 44 mm membrane holder (which has been modified toinclude additional holes to promote better filtration). An enclosingfilm is visible adhered to the top of the stirred cell unit. The sprayhead holder containing the LB-100 has been inserted into the chamber toa distance of 82 mm from the emitter tip to the surface of the filtermembrane.

FIG. 133 is a plot showing data characterizing the delivery efficiencyand viability using the LB-100 nebulizer. Graph demonstrates averagedelivery efficiency of 59.63%±1.2 and average viability data of74.6%±5.3 across 3 technical repeats. The cell monolayer was formedusing a 0.4 μm PCTE filter membrane and 20×10⁷ cells and application of120 mbar pressure for 40 s. The LB-100 spray parameters were 2.5 barpressure; 600 mbar 400 ms spray duration (100 μl volume) and 82 mmdistance. The data represents results from three experimentscharacterizing the delivery efficiency and viability.

FIG. 134 is an embodiment of an example delivery system for clinicaluse.

DETAILED DESCRIPTION

Difficulty in transfecting molecules into non-adherent cells has plaguedresearch and therapeutic, e.g., cell therapy, gene therapy, geneticalteration, for decades. A reason for the difficulty in transfectingsuch cells may be that non-adherent cells lack cell surface heparansulfate proteoglycans, molecules are responsible for adhesion of cellsto the extra-cellular matrix. Transfection methods such aselectroporation and/or nucleofections have drawbacks in that theycompromise the viability of cells, the ability of the cells to resumeproliferation after treatment, and the function of the cells, e.g.,immune activity of lymphocytes. The transfection compositions andmethods described herein (Soluporation) do not have such drawbacks andtherefore are characterized as having significant advantages overearlier methods of introducing cargo molecules into mammalian cells,e.g., difficult-to-transfect non-adherent/suspension cells.

The invention is based on the surprising discovery that compounds ormixtures of compounds (compositions) are delivered into the cytoplasm ofeukaryotic cells by contacting the cells with a solution containing acompound(s) to be delivered (e.g., payload) and an agent that reversiblypermeates or dissolves a cell membrane. Preferably, the solution isdelivered to the cells in the form of a spray, e.g., aqueous particles.(see, e.g., PCT/US2015/057247 and PCT/IB2016/001895, hereby incorporatedin their entirety by reference). For example, the cells are coated withthe spray but not soaked or submersed in the deliverycompound-containing solution. Exemplary agents that permeate or dissolvea eukaryotic cell membrane include alcohols and detergents such asethanol and Triton X-100, respectively. Other exemplary detergents,e.g., surfactants include polysorbate 20 (e.g., Tween 20),3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), sodium dodecyl sulfate (SDS), and octyl glucoside.

An example of conditions to achieve a coating of a population of coatedcells include delivery of a fine particle spray, e.g., the conditionsexclude dropping or pipetting a bolus volume of solution on the cellssuch that a substantial population of the cells are soaked or submergedby the volume of fluid. Thus, the mist or spray comprises a ratio ofvolume of fluid to cell volume. Alternatively, the conditions comprise aratio of volume of mist or spray to exposed cell area, e.g., area ofcell membrane that is exposed when the cells exist as a confluent orsubstantially confluent layer on a substantially flat surface such asthe bottom of a tissue culture vessel, e.g., a well of a tissue cultureplate, e.g., a microtiter tissue culture plate.

“Cargo” or “payload” are terms used to describe a compound, orcomposition that is delivered via an aqueous solution across a cellplasma membrane and into the interior of a cell.

In an aspect, delivering a payload across a plasma membrane of a cellincludes providing a population of cells and contacting the populationof cells with a volume of an aqueous solution. The aqueous solutionincludes the payload and an alcohol content greater than 5 percentconcentration. The volume of the aqueous solution may be a function ofexposed surface area of the population of cells, or may be a function ofa number of cells in the population of cells.

In another aspect, a composition for delivering a payload across aplasma membrane of a cell includes an aqueous solution including thepayload, an alcohol at greater than 5 percent concentration, greaterthan 46 mM salt, less than 121 mM sugar, and less than 19 mM bufferingagent. For example, the alcohol, e.g., ethanol, concentration does notexceed 50%.

One or more of the following features can be included in any feasiblecombination. The volume of solution to be delivered to the cells is aplurality of units, e.g., a spray, e.g., a plurality of droplets onaqueous particles. The volume is described relative to an individualcell or relative to the exposed surface area of a confluent orsubstantially confluent (e.g., at least 75%, at least 80% confluent,e.g., 85%, 90%, 95%, 97%, 98%, 100%) cell population. For example, thevolume can be between 6.0×10⁻⁷ microliter per cell and 7.4×10⁻⁴microliter per cell. The volume is between 4.9×10⁻⁶ microliter per celland 2.2×10⁻³ microliter per cell. The volume can be between 9.3×10−6microliter per cell and 2.8×10⁻⁵ microliter per cell. The volume can beabout 1.9×10⁻⁵ microliters per cell, and about is within 10 percent. Thevolume is between 6.0×10⁻⁷ microliter per cell and 2.2×10⁻³ microliterper cell. The volume can be between 2.6×10⁻⁹ microliter per squaremicrometer of exposed surface area and 1.1×10⁻⁶ microliter per squaremicrometer of exposed surface area. The volume can be between 5.3×10-8microliter per square micrometer of exposed surface area and 1.6×10⁻⁷microliter per square micrometer of exposed surface area. The volume canbe about 1.1×10⁻⁷ microliter per square micrometer of exposed surfacearea. About can be within 10 percent.

Confluency of cells refers to cells in contact with one another on asurface. For example, it can be expressed as an estimated (or counted)percentage, e.g., 10% confluency means that 10% of the surface, e.g., ofa tissue culture vessel, is covered with cells, 100% means that it isentirely covered. For example, adherent cells grow two dimensionally onthe surface of a tissue culture well, plate or flask. Non-adherent cellscan be spun down, pulled down by a vacuum, or tissue culture mediumaspiration off the top of the cell population, or removed by aspirationor vacuum removal from the bottom of the vessel.

Contacting the population of cells with the volume of aqueous solutioncan be performed by gas propelling the aqueous solution to form a spray.The gas can include nitrogen, ambient air, or an inert gas. The spraycan include discrete units of volume ranging in size from, lnm to 100μm, e.g., 30-100 μm in diameter. The spray includes discrete units ofvolume with a diameter of about 30-50 μm. A total volume of aqueoussolution of 20 μl can be delivered in a spray to a cell-occupied area ofabout 1.9 cm², e.g., one well of a 24-well culture plate. A total volumeof aqueous solution of 10 μl is delivered to a cell-occupied area ofabout 0.95 cm², e.g., one well of a 48-well culture plate. Typically,the aqueous solution includes a payload to be delivered across a cellmembrane and into cell, and the second volume is a buffer or culturemedium that does not contain the payload. Alternatively, the secondvolume (buffer or media) can also contain payload. In some embodiments,the aqueous solution includes a payload and an alcohol, and the secondvolume does not contain alcohol (and optionally does not containpayload). The population of cells can be in contact with said aqueoussolution for 0.1 10 minutes prior to adding a second volume of buffer orculture medium to submerse or suspend said population of cells. Thebuffer or culture medium can be phosphate buffered saline (PBS). Thepopulation of cells can be in contact with the aqueous solution for 2seconds to 5 minutes prior to adding a second volume of buffer orculture medium to submerse or suspend the population of cells. Thepopulation of cells can be in contact with the aqueous solution, e.g.,containing the payload, for 30 seconds to 2 minutes prior to adding asecond volume of buffer or culture medium, e.g., without the payload, tosubmerse or suspend the population of cells. The population of cells canbe in contact with a spray for about 1-2 minutes prior to adding thesecond volume of buffer or culture medium to submerse or suspend thepopulation of cells. During the time between spraying of cells andaddition of buffer or culture medium, the cells remain hydrated by thelayer of moisture from the spray volume.

The aqueous solution can include an ethanol concentration of 5 to 30%.The aqueous solution can include one or more of 75 to 98% H₂O, 2 to 45%ethanol, 6 to 91 mM sucrose, 2 to 500 mM KCl, 2 to 35 mM ammoniumacetate, and 1 to 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) (HEPES). For example, the delivery solution contains 106 mM KCland 27% ethanol.

The population of cells can include adherent cells or non-adherentcells. The adherent cells can include at least one of primarymesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells,neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinesehamster ovary (CHO) cells, and human embryonic kidney (HEK) cells orimmortalized cells, such as cell lines. In preferred embodiments, thepopulation of cells comprises non-adherent cells, e.g., the %non-adherent cells in the population is at least 50%, 60%, 75%, 80%,90%, 95%, 98%, 99% or 100% non-adherent cells. Non-adherent cellsprimary cells as well as immortalized cells (e.g., cells of a cellline). Exemplary non-adherent/suspension cells include primaryhematopoietic stem cell (HSC), T cells (e.g., CD3+ cells, CD4+ cells,CD8+ cells), natural killer (NK) cells, cytokine-induced killer (CIK)cells, human cord blood CD34+ cells, B cells, or cell lines such asJurkat T cell line.

The payload can include a small chemical molecule, a peptide or protein,or a nucleic acid. The small chemical molecule can be less than 1,000Da. The chemical molecule can include MitoTracker® Red CMXRos, propidiumiodide, methotrexate, and/or DAPI (4′,6-diamidino-2-phenylindole). Thepeptide can be about 5,000 Da. The peptide can include ecallantide undertrade name Kalbitor, is a 60 amino acid polypeptide for the treatment ofhereditary angioedema and in prevention of blood loss in cardiothoracicsurgery), Liraglutide (marketed as the brand name Victoza, is used forthe treatment of type II diabetes, and Saxenda for the treatment ofobesity), and Icatibant (trade name Firazyer, a peptidomimetic for thetreatment of acute attacks of hereditary angioedema). Thesmall-interfering ribonucleic acid (siRNA) molecule can be about 20-25base pairs in length, or can be about 10,000-15,000 Da. The siRNAmolecule can reduces the expression of any gene product, e.g., knockdownof gene expression of clinically relevant target genes or of modelgenes, e.g., glyceraldehyde-3phosphate dehydrogenase (GAPDH) siRNA,GAPDH siRNA-FITC, cyclophilin B siRNA, and/or lamin siRNA. Proteintherapeutics can include peptides, enzymes, structural proteins,receptors, cellular proteins, or circulating proteins, or fragmentsthereof. The protein or polypeptide be about 100-500,000 Da, e.g.,1,000-150,000 Da. The protein can include any therapeutic, diagnostic,or research protein or peptide, e.g., beta-lactoglobulin, ovalbumin,bovine serum albumin (BSA), and/or horseradish peroxidase. In otherexamples, the protein can include a cancer-specific apoptotic protein,e.g., Tumor necrosis factor-related apoptosis inducing protein (TRAIL).

An antibody is generally be about 150,000 Da in molecular mass. Theantibody can include an anti-actin antibody, an anti-GAPDH antibody, ananti-Src antibody, an anti-Myc ab, and/or an anti-Raf antibody. Theantibody can include a green fluorescent protein (GFP) plasmid, a GLucplasmid and, and a BATEM plasmid. The DNA molecule can be greater than5,000,000 Da. In some examples, the antibody can be a murine-derivedmonoclonal antibody, e.g., ibritumomab tiuxetin, muromomab-CD3,tositumomab, a human antibody, or a humanized mouse (or other species oforigin) antibody. In other examples, the antibody can be a chimericmonoclonal antibody, e.g., abciximab, basiliximab, cetuximab,infliximab, or rituximab. In still other examples, the antibody can be ahumanized monoclonal antibody, e.g., alemtuzamab, bevacizumab,certolizumab pegol, daclizumab, gentuzumab ozogamicin, trastuzumab,tocilizumab, ipilimumamb, or panitumumab. The antibody can comprise anantibody fragment, e.g., abatecept, aflibercept, alefacept, oretanercept. The invention encompasses not only an intact monoclonalantibody, but also an immunologically-active antibody fragment, e. g. ,a Fab or (Fab)2 fragment; an engineered single chain Fv molecule; or achimeric molecule, e.g., an antibody which contains the bindingspecificity of one antibody, e.g., of murine origin, and the remainingportions of another antibody, e.g., of human origin.

The payload can include a therapeutic agent. A therapeutic agent, e.g.,a drug, or an active agent”, can mean any compound useful fortherapeutic or diagnostic purposes, the term can be understood to meanany compound that is administered to a patient for the treatment of acondition. Accordingly, a therapeutic agent can include, proteins,peptides, antibodies, antibody fragments, and small molecules.Therapeutic agents described in U.S. Pat. No.7,667,004 (incorporatedherein by reference) can be used in the methods described herein. Thetherapeutic agent can include at least one of cisplatin, aspirin,statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin,rosuvastatin, simvastatin, promazine HCl, chloropromazine HCl,thioridazine HCl, Polymyxin B sulfate, chloroxine, benfluorex HCl andphenazopyridine HCl), and fluoxetine. The payload can include adiagnostic agent. The diagnostic agent can include a detectable label ormarker such as at least one of methylene blue, patent blue V, andindocyanine green. The payload can include a fluorescent molecule. Thepayload can include a detectable nanoparticle. The nanoparticle caninclude a quantum dot.

The population of non-adherent cells can be substantially confluent,such as greater than 75 percent confluent. Confluency of cells refers tocells in contact with one another on a surface. For example, it can beexpressed as an estimated (or counted) percentage, e.g., 10% confluencymeans that 10% of the surface, e.g., of a tissue culture vessel, iscovered with cells, 100% means that it is entirely covered. For example,adherent cells grow two dimensionally on the surface of a tissue culturewell, plate or flask. Non-adherent cells can be spun down, pulled downby a vacuum, or tissue culture medium aspiration off the top of the cellpopulation, or removed by aspiration or vacuum removal from the bottomof the vessel. The population of cells can form a monolayer of cells.

The alcohol can be selected from methanol, ethanol, isopropyl alcohol,butanol and benzyl alcohol. The salt can be selected from NaCl, KCl,Na₂HPO₄, KH₂PO₄, and C₂H₃O₂NH. In preferred embodiments, the salt isKCl. The sugar can include sucrose. The buffering agent can include4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid.

The present subject matter relates to a method for delivering moleculesacross a plasma membrane. The present subject matter finds utility inthe field of intra-cellular delivery, and has application in, forexample, delivery of molecular biological and pharmacologicaltherapeutic agents to a target site, such as a cell, tissue, or organ.The method of the present subject matter comprises introducing themolecule to an aqueous composition to form a matrix; atomizing thematrix into a spray; and contacting the matrix with a plasma membrane.

This present subject matter relates to a composition for use indelivering molecules across a plasma membrane. The present subjectmatter finds utility in the field of intra-cellular delivery, and hasapplication in, for example, delivery of molecular biological andpharmacological therapeutic agents to a target site, such as a cell,tissue, or organ. The composition of the present subject mattercomprises an alcohol; a salt; a sugar; and/or a buffering agent.

In some implementations, demonstrated is a permeabilisation techniquethat facilitates intracellular delivery of molecules independent of themolecule and cell type. Nanoparticles, small molecules, nucleic acids,proteins and other molecules can be efficiently delivered intosuspension cells or adherent cells in situ, including primary cells andstem cells, with low cell toxicity and the technique is compatible withhigh throughput and automated cell-based assays.

The example methods described herein include a payload, wherein thepayload includes an alcohol. By the term “an alcohol” is meant apolyatomic organic compound including a hydroxyl (—OH) functional groupattached to at least one carbon atom. The alcohol may be a monohydricalcohol and may include at least one carbon atom, for example methanol.The alcohol may include at least two carbon atoms (e.g. ethanol). Inother aspects, the alcohol comprises at least three carbons (e.g.isopropyl alcohol). The alcohol may include at least four carbon atoms(e.g., butanol), or at least seven carbon atoms (e.g., benzyl alcohol).The example payload may include no more than 50% (v/v) of the alcohol,more preferably, the payload comprises 2-45% (v/v) of the alcohol, 5-40%of the alcohol, and 10-40% of the alcohol. The payload may include20-30% (v/v) of the alcohol.

Most preferably, the payload delivery solution includes 25% (v/v) of thealcohol. Alternatively, the payload can include 2-8% (v/v) of thealcohol, or 2% of the alcohol. The alcohol may include ethanol and thepayload comprises 5, 10, 20, 25, 30, and up to 40% or 50% (v/v) ofethanol, e.g., 27%. Example methods may include methanol as the alcohol,and the payload may include 5, 10, 20, 25, 30, or 40% (v/v) of themethanol. The payload may include 2-45% (v/v) of methanol, 20-30% (v/v),or 25% (v/v) methanol. Preferably, the payload includes 20-30% (v/v) ofmethanol. Further alternatively, the alcohol is butanol and the payloadcomprises 2, 4, or 8% (v/v) of the butanol.

In some aspects of the present subject matter, the payload is in anisotonic solution or buffer.

According to the present subject matter, the payload may include atleast one salt. The salt may be selected from NaCl, KCl, Na₂HPO₄,C₂H₃O₂NH₄ and KH₂PO₄. For example, KCl concentration ranges from 2 mM to500 mM. In some preferred embodiments, the concentration is greater than100 mM, e.g., 106 mM.

According to example methods of the present subject matter, the payloadmay include a sugar (e.g., a sucrose, or a disaccharide). According toexample methods, the payload comprises less than 121 mM sugar, 6-91 mM,or 26-39 mM sugar. Still further, the payload includes 32 mM sugar(e.g., sucrose). Optionally, the sugar is sucrose and the payloadcomprises 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mM sucrose.

According to example methods of the present subject matter, the payloadmay include a buffering agent (e.g. a weak acid or a weak base). Thebuffering agent may include a zwitterion. According to example methods,the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid. The payload may comprise less than 19 mM buffering agent (e.g.,1-15 mM, or 4-6 mM or 5 mM buffering agent). According to examplemethods, the buffering agent is4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and the payloadcomprises 1, 2, 3, 4, 5, 10, 12, 14 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Further preferably,the payload comprises 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid.

According to example methods of the present subject matter, the payloadincludes ammonium acetate. The payload may include less than 46 mMammonium acetate (e.g., between 2-35 mM, 10-15 mM, ore 12 mM ammoniumacetate). The payload may include 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or33.6 mM ammonium acetate.

The volume of aqueous solution performed by gas propelling the aqueoussolution may include compressed air (e.g. ambient air), otherimplementations may include inert gases, for example, helium, neon, andargon.

In certain aspects of the present subject matter, the population ofcells may include adherent cells (e.g., lung, kidney, immune cells suchas macrophages) or non-adherent cells (e.g., suspension cells).

In certain aspects of the present subject matter, the population ofcells may be substantially confluent, and substantially may includegreater than 75 percent confluent. In preferred implementations, thepopulation of cells may form a single monolayer.

According to example methods, the payload to be delivered has an averagemolecular weight of up to 20,000,000 Da. In some examples, the payloadto be delivered can have an average molecular weight of up to 2,000,000Da. In some implementations, the payload to be delivered may have anaverage molecular weight of up to 150,000 Da. In furtherimplementations, the payload to be delivered has an average molecularweight of up to 15,000 Da, 5,000 Da or 1,000 Da.

The payload to be delivered across the plasma membrane of a cell mayinclude a small chemical molecule, a peptide or protein, apolysaccharide or a nucleic acid or a nanoparticle. A small chemicalmolecule may be less than 1,000 Da, peptides may have molecular weightsabout 5,000 Da, siRNA may have molecular weights around 15,000 Da,antibodies may have molecular weights of about 150,000 Da and DNA mayhave molecular weights of greater than or equal to 5,000,000 Da. Inpreferred embodiments, the payload comprises mRNA.

According to example methods, the payload includes 3.0-150.0 μM of amolecule to be delivered, more preferably, 6.6-150.0 μM molecule to bedelivered (e.g. 3.0, 3.3, 6.6, or 150.0 μM molecule to be delivered). Insome implementations, the payload to be delivered has an averagemolecular weight of up to 15,000 Da, and the payload includes 3.3 μMmolecules to be delivered.

According to example methods, the payload to be delivered has an averagemolecular weight of up to 15,000 Da, and the payload includes 6.6 μM tobe delivered. In some implementations, the payload to be delivered hasan average molecular weight of up to 1,000 Da, and the payload includes150.0 μM to be delivered.

According to further aspects of the present subject matter, a method fordelivering molecules of more than one molecular weight across a plasmamembrane is provided; the method including the steps of: introducing themolecules of more than one molecular weight to an aqueous solution; andcontacting the aqueous solution with a plasma membrane.

In some implementations, the method includes introducing a firstmolecule having a first molecular weight and a second molecule having asecond molecular weight to the payload, wherein the first and secondmolecules may have different molecular weights, or wherein, the firstand second molecules may have the same molecular weights. According toexample methods, the first and second molecules may be differentmolecules.

In some implementations, the payload to be delivered may include atherapeutic agent, or a diagnostic agent, including, for example,cisplatin, aspirin, various statins (e.g., pitavastatin, atorvastatin,lovastatin, pravastatin, rosuvastatin, simvastatin, promazine HCl,chloropromazine HCl, thioridazine HCl, Polymyxin B sulfate, chloroxine,benfluorex HCl and phenazopyridine HCl), and fluoxetine. Othertherapeutic agents include antimicrobials (aminoclyclosides (e.g.gentamicin, neomycin, streptomycin), penicillins (e.g., amoxicillin,ampicillin), glycopeptides (e.g., avoparcin, vancomycin), macrolides(e.g., erythromycin, tilmicosin, tylosin), quinolones (e.g.,sarafloxacin, enrofloxin), streptogramins (e.g., viginiamycin,quinupristin-dalfoprisitin), carbapenems, lipopeptides, oxazolidinones,cycloserine, ethambutol, ethionamide, isoniazrid, para-aminosalicyclicacid, and pyrazinamide). In some examples, an anti-viral (e.g.,Abacavir, Aciclovir, Enfuvirtide, Entecavir, Nelfinavir, Nevirapine,Nexavir, Oseltamivir Raltegravir, Ritonavir, Stavudine, andValaciclovir). The therapeutic may include a protein-based therapy forthe treatment of various diseases, e.g., cancer, infectious diseases,hemophilia, anemia, multiple sclerosis, and hepatitis B or C.

Additional exemplary payloads can also include detectable markers orlabels such as methylene blue, Patent blue V, and Indocyanine green.

The methods described herein may also include the payload including of adetectable moiety, or a detectable nanoparticle (e.g., a quantum dot).The detectable moiety may include a fluorescent molecule or aradioactive agent (e.g., ¹²⁵I). When the fluorescent molecule is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, p-phthaldehyde and fluorescamine Themolecule can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the molecule using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA). The molecule also can be detectably labeled by coupling itto a chemiluminescent compound. The presence of thechemiluminescent-tagged molecule is then determined by detecting thepresence of luminescence that arises during the course of chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

In additional embodiments, the payload to be delivered may include acomposition that edits genomic DNA (i.e., gene editing tools). Forexample, the gene editing composition may include a compound or complexthat cleaves, nicks, splices, rearranges, translocates, recombines, orotherwise alters genomic DNA. Alternatively or in addition, a geneediting composition may include a compound that (i) may be included agene-editing complex that cleaves, nicks, splices, rearranges,translocates, recombines, or otherwise alters genomic DNA; or (ii) maybe processed or altered to be a compound that is included in agene-editing complex that cleaves, nicks, splices, rearranges,translocates, recombines, or otherwise alters genomic DNA. In variousembodiments, the gene editing composition comprises one or more of (a)gene editing protein; (b) RNA molecule; and/or (c) ribonucleoprotein(RNP).

In some embodiments, the gene editing composition comprises a geneediting protein, and the gene editing protein is a zinc finger nuclease(ZFN), a transcription activator-like effector nuclease (TALEN), a Casprotein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. Inadditional embodiments, the gene editing protein may be a fusionproteins that combine homing endonucleases with the modular DNA bindingdomains of TALENs (megaTAL). For example, megaTAL may be delivered as aprotein or alternatively, a mRNA encoding a megaTAL protein is deliveredto the cells.

In various embodiments, the gene editing composition comprises a RNAmolecule, and the RNA molecule comprises a sgRNA, a crRNA, and/or atracrRNA.

In certain embodiments, the gene editing composition comprises a RNP,and the RNP comprises a Cas protein and a sgRNA or a crRNA and atracrRNA. Aspects of the present subject matter are particularly usefulfor controlling when and for how long a particular gene-editing compoundis present in a cell.

In various implementations of the present subject matter, the geneediting composition is detectable in a population of cells, or theprogeny thereof, for (a) about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,24, 48, 60, 72, 0.5-2, 0.5-6, 6-12 or 0.5-72 hours after the populationof cells is contacted with the aqueous solution, or (b) less than about0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6,6-12 or 0.5-72 hours after the population of cells is contacted with theaqueous solution.

In some embodiments, the genome of cells in the population of cells, orthe progeny thereof, comprises at least one site-specific recombinationsite for the Cre recombinase, Hin recombinase, or Flp recombinase.

Aspects of the present invention relate to cells that comprise one geneediting compound, and inserting another gene editing compound into thecells. For example, one component of an RNP could be introduced intocells that express or otherwise already contain another component of theRNP. For example, cells in a population of cells, or the progenythereof, may comprise a sgRNA, a crRNA, and/or a tracrRNA. In someembodiments the population of cells, or the progeny thereof, expressesthe sgRNA, crRNA, and/or tracrRNA. Alternatively or in addition, cellsin a population of cells, or the progeny thereof, express a Cas protein.

Various implementations of the subject matter herein include a Casprotein. In some embodiments, the Cas protein is a Cas9 protein or amutant thereof. Exemplary Cas proteins (including Cas9 and non-limitingexamples of Cas9 mutants) are described herein.

In various aspects, the concentration of Cas9 protein may range fromabout 0.1 to about 25 μg. For example, the concentration of Cas9 may beabout 1 μg, about 5 μg, about 10 μg, about 15 μg, or about 20 μg.Alternatively, the concentration of Cas9 may range from about 10 ng/μLto about 300 ng/μL; for example from about 10 ng/μL to about 200 ng/μl;or from about 10 ng/μL to about 100 ng/μl, or from about 10 ng/μL toabout 50 ng/μl.

In certain embodiments, the gene editing composition comprises (a) afirst sgRNA molecule and a second sgRNA molecule, wherein the nucleicacid sequence of the first sgRNA molecule is different from the nucleicacid sequence of the second sgRNA molecule; (b) a first RNP comprising afirst sgRNA and a second RNP comprising a second sgRNA, wherein thenucleic acid sequence of the first sgRNA molecule is different from thenucleic acid sequence of the second sgRNA molecule; (c) a first crRNAmolecule and a second crRNA molecule, wherein the nucleic acid sequenceof the first crRNA molecule is different from the nucleic acid sequenceof the second crRNA molecule; (d) a first crRNA molecule and a secondcrRNA molecule, wherein the nucleic acid sequence of the first crRNAmolecule is different from the nucleic acid sequence of the second crRNAmolecule, and further comprising a tracrRNA molecule; or (e) a first RNPcomprising a first crRNA and a tracrRNA and a second RNP comprising asecond crRNA and a tracrRNA, wherein the nucleic acid sequence of thefirst crRNA molecule is different from the nucleic acid sequence of thesecond crRNA molecule.

In aspects, the ratio of the Cas9 protein to guide RNA may be 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

In embodiments, increasing the number of times that cells go through thedelivery process (alternatively, increasing the number of doses), mayincrease the percentage edit; wherein, in some embodiments the number ofdoses may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

In various embodiments, the first and second sgRNA or first and secondcrRNA molecules together comprise nucleic acid sequences complementaryto target sequences flanking a gene, an exon, an intron, anextrachromosomal sequence, or a genomic nucleic acid sequence, whereinthe gene, an exon, intron, extrachromosomal sequence, or genomic nucleicacid sequence is about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 1-100, kilobases in length or is at least about 1, 2, 3, 4, 5,6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100, kilobases in length.In some embodiments, the use of pairs of RNPs comprising the first andsecond sgRNA or first and second crRNA molecules may be used to create apolynucleotide molecule comprising the gene, exon, intron,extrachromosomal sequence, or genomic nucleic acid sequence.

In certain embodiments, the target sequence of a sgRNA or crRNA is about12 to about 25, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 17-23, or 18-22, nucleotides long. In some embodiments, thetarget sequence is 20 nucleotides long or about 20 nucleotides long.

In various embodiments, the first and second sgRNA or first and secondcrRNA molecules are complementary to sequences flanking anextrachromosomal sequence that is within an expression vector.

Aspects of the present subject matter relate to the delivery of multiplecomponents of a gene-editing complex, where the multiple components arenot complexed together. In some embodiments, gene editing compositioncomprises at least one gene editing protein and at least one nucleicacid, wherein the gene editing protein and the nucleic acid are notbound to or complexed with each other.

The present subject matter allows for high gene editing efficiency whilemaintaining high cell viability. In some embodiments, at least about 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of thepopulation of cells, or the progeny thereof, become genetically modifiedafter contact with the aqueous solution. In various embodiments, atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or moreof the population of cells, or the progeny thereof, are viable aftercontact with the aqueous solution.

In certain embodiments, the gene editing composition inducessingle-strand or double-strand breaks in DNA within the cells. In someembodiments the gene editing composition further comprises a repairtemplate polynucleotide. In various embodiments, the repair templatecomprises (a) a first flanking region comprising nucleotides in asequence complementary to about 40 to about 90 base pairs on one side ofthe single or double strand break and a second flanking regioncomprising nucleotides in a sequence complementary to about 40 to about90 base pairs on the other side of the single or double strand break; or(b) a first flanking region comprising nucleotides in a sequencecomplementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,or 90 base pairs on one side of the single or double strand break and asecond flanking region comprising nucleotides in a sequencecomplementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,or 90 base pairs on the other side of the single or double strand break.Non-limiting descriptions relating to gene editing (including repairtemplates) using the CRISPR-Cas system are discussed in Ran et al.(2013) Nat Protoc. 2013 November; 8(11): 2281-2308, the entire contentof which is incorporated herein by reference. Embodiments involvingrepair templates are not limited to those comprising the CRISPR-Cassystem.

In various implementations of the present subject matter, the volume ofaqueous solution is delivered to the population of cells in the form ofa spray. In some embodiments, the volume is between 6.0×10⁻⁷ microliterper cell and 7.4×10⁻⁴ microliter per cell. In certain embodiments, thespray comprises a colloidal or sub-particle comprising a diameter of 10nm to 100 μm. In various embodiments, the volume is between 2.6×10⁻⁹microliter per square micrometer of exposed surface area and 1.1×10⁻⁶microliter per square micrometer of exposed surface area.

In some embodiments, the RNP has a size of approximately 100 Å×100 Å×50Å or 10 nm×10 nm×5 nm. In various embodiments, the size of sprayparticles is adjusted to accommodate at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more RNPs per spray particle.

For example, contacting the population of cells with the volume ofaqueous solution may be performed by gas propelling the aqueous solutionto form a spray. In certain embodiments, the population of cells is incontact with said aqueous solution for 0.01-10 minutes (e.g., 0.1 10minutes) prior to adding a second volume of buffer or culture medium tosubmerse or suspend said population of cells.

In various embodiments, the population of cells includes at least one ofprimary or immortalized cells. For example, the population of cells mayinclude mesenchymal stem cells, lung cells, neuronal cells, fibroblasts,human umbilical vein (HUVEC) cells, and human embryonic kidney (HEK)cells, primary or immortalized hematopoietic stem cell (HSC), T cells,natural killer (NK) cells, cytokine-induced killer (CIK) cells, humancord blood CD34+ cells, B cells. Non limiting examples of T cells mayinclude CD8+ or CD4+ T cells. In some aspects, the CD8+ subpopulation ofthe CD3+ T cells are used. CD8⁺ T cells may be purified from the PBMCpopulation by positive isolation using anti-CD8 beads. In some aspectsprimary NK cells are isolated from PBMCs and GFP mRNA may be deliveredby platform delivery technology (i.e., 3% expression and 96% viabilityat 24 hours). In additional aspects, NK cell lines, e.g., NK92 may beused.

Cell types also include cells that have previously been modified forexample T cells, NK cells and MSC to enhance their therapeutic efficacy.For example: T cells or NK cells that express chimeric antigen receptors(CAR T cells, CAR NK cells, respectively); T cells that express modifiedT cell receptor (TCR); MSC that are modified virally or non-virally tooverexpress therapeutic proteins that complement their innate properties(e.g. delivery of Epo using lentiviral vectors or BMP-2 using AAV-6)(reviewed in Park et al, Methods, 2015 August; 84-16.); MSC that areprimed with non-peptidic drugs or magnetic nanoparticles for enhancedefficacy and externally regulated targeting respectively (Park et al.,2015); MSC that are functionalised with targeting moieties to augmenttheir homing toward therapeutic sites using enzymatic modification (e.g.Fucosyltransferase), chemical conjugation (eg. modification of SLeX onMSC by using N-hydroxy-succinimide (NHS) chemistry) or non-covalentinteractions (eg. engineering the cell surface with palmitated proteinswhich act as hydrophobic anchors for subsequent conjugation ofantibodies) (Park et al., 2015). For example, T cells, e.g., primary Tcells or T cell lines, that have been modified to express chimericantigen receptors (CAR T cells) may further be treated according to theinvention with gene editing proteins and or complexes containing guidenucleic acids specific for the CAR encoding sequences for the purpose ofediting the gene(s) encoding the CAR, thereby reducing or stopping theexpression of the CAR in the modified T cells.

Aspects of the present invention relate to the expression vector-freedelivery of gene editing compounds and complexes to cells and tissues,such as delivery of Cas-gRNA ribonucleoproteins for genome editing inprimary human T cells, hematopoietic stem cells (HSC), and mesenchymalstromal cells (MSC). In some example, mRNA encoding such proteins aredelivered to the cells.

Various aspects of the CRISPR-Cas system are known in the art.Non-limiting aspects of this system are described, e.g., in U.S. Pat.No. 9,023,649, issued May 5, 2015; U.S. Pat. No. 9,074,199, issued Jul.7, 2015; U.S. Pat. No. 8,697,359, issued Apr. 15, 2014; U.S. Pat. No.8,932,814, issued Jan. 13, 2015; PCT International Patent ApplicationPublication No. WO 2015/071474, published Aug. 27, 2015; Cho et al.,(2013) Nature Biotechnology Vol 31 No 3 pp 230-232 (includingsupplementary information); and Jinek et al., (2012) Science Vol 337 No6096 pp 816-821, the entire contents of each of which are incorporatedherein by reference.

Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,homologs thereof, or modified versions thereof. These enzymes are known;for example, the amino acid sequence of S. pyogenes Cas9 protein may befound in the SwissProt database under accession number Q99ZW2 and in theNCBI database as under accession number Q99ZW2.1. UniProt databaseaccession numbers A0A0G4DEU5 and CDJ55032 provide another example of aCas9 protein amino acid sequence. Another non-limiting example is aStreptococcus thermophilus Cas9 protein, the amino acid sequence ofwhich may be found in the UniProt database under accession numberQ03JI6.1. In some embodiments, the unmodified CRISPR enzyme has DNAcleavage activity, such as Cas9. In certain embodiments the CRISPRenzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. Invarious embodiments, the CRISPR enzyme directs cleavage of one or bothstrands at the location of a target sequence, such as within the targetsequence and/or within the complement of the target sequence. In someembodiments, the CRISPR enzyme directs cleavage of one or both strandswithin about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200,500, or more base pairs from the first or last nucleotide of a targetsequence. In some embodiments, a vector encodes a CRISPR enzyme that ismutated to with respect to a corresponding wild-type enzyme such thatthe mutated CRISPR enzyme lacks the ability to cleave one or bothstrands of a target polynucleotide containing a target sequence. Forexample, an aspartate-to-alanine substitution in the RuvC I catalyticdomain of Cas9 from S. pyogenes converts Cas9 from a nuclease thatcleaves both strands to a nickase (cleaves a single strand). Otherexamples of mutations that render Cas9 a nickase include, withoutlimitation, H840A, N854A, and N863A. In aspects of the invention,nickases may be used for genome editing via homologous recombination.

In certain embodiments, a Cas9 nickase may be used in combination withguide sequence(s), e.g., two guide sequences, which target respectivelysense and antisense strands of the DNA target. This combination allowsboth strands to be nicked and used to induce NHEJ.

As a further example, two or more catalytic domains of Cas9 (RuvC I,RuvC II, and RuvC III) may be mutated to produce a mutated Cas9substantially lacking all DNA cleavage activity. A D10A mutation may becombined with one or more of H840A, N854A, or N863A mutations to producea Cas9 enzyme substantially lacking all DNA cleavage activity. Incertain embodiments, a CRISPR enzyme is considered to substantially lackall DNA cleavage activity when the DNA cleavage activity of the mutatedenzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower withrespect to its non-mutated form. Other mutations may be useful; wherethe Cas9 or other CRISPR enzyme is from a species other than S.pyogenes, mutations in corresponding amino acids may be made to achievesimilar effects.

In certain embodiments, a protein being delivered (such as a Cas proteinor a variant thereof) may include a subcellular localization signal. Forexample, the Cas protein within a RNP may comprise a subcellularlocalization signal. Depending on context, a fusion protein comprising,e.g., Cas9 and a nuclear localization signal may be referred to as“Cas9” herein without specifying the inclusion of the nuclearlocalization signal. In some embodiments, the payload (such as an RNP)comprises a fusion-protein that comprises a localization signal. Forexample, the fusion-protein may contain a nuclear localization signal, anucleolar localization signal, or a mitochondrial targeting signal. Suchsignals are known in the art, and non-limiting examples are described inKalderon et al., (1984) Cell 39 (3 Pt 2): 499-509; Makkerh et al.,(1996) Curr Biol. 6 (8):1025-7; Dingwall et al., (1991) Trends inBiochemical Sciences 16 (12): 478-81; Scott et al., (2011) BMCBioinformatics 12:317 (7 pages); Omura T (1998) J Biochem.123(6):1010-6; Rapaport D (2003) EMBO Rep. 4(10):948-52; and Brocard &Hartig (2006) Biochimica et Biophysica Acta (BBA)—Molecular CellResearch 1763(12):1565-1573, the contents of each of which are herebyincorporated herein by reference. In various embodiments, the Casprotein may comprise more than one localization signals, such as 2, 3,4, 5, or more nuclear localization signals. In some embodiments, thelocalization signal is at the N-terminal end of the Cas protein and inother embodiments the localization signal is at the C-terminal end ofthe Cas protein.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzymeis codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, mouse, rat, rabbit, dog, or non-human primate. In general, codonoptimization refers to a process of modifying a nucleic acid sequencefor enhanced expression in the host cells of interest by replacing atleast one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15,20, 25, 50, or more codons) of the native sequence with codons that aremore frequently or most frequently used in the genes of that host cellwhile maintaining the native amino acid sequence. Various speciesexhibit particular bias for certain codons of a particular amino acid.Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis.

Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database”, and thesetables can be adapted in a number of ways. See Nakamura, Y., et al.“Codon usage tabulated from the international DNA sequence databases:status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computeralgorithms for codon optimizing a particular sequence for expression ina particular host cell are also available, such as Gene Forge (Aptagen;Jacobus, Pa.), are also available. In some embodiments, one or morecodons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons)in a sequence encoding a CRISPR enzyme corresponding to the mostfrequently used codon for a particular amino acid.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. In some embodiments, the degree ofcomplementarity is 100%. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). In some embodiments, a guide sequence is about ormore than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotidesin length. In certain embodiments, a guide sequence is less than about75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.The ability of a guide sequence to direct sequence-specific binding of aCRISPR complex to a target sequence may be assessed by any suitableassay. For example, the components of a CRISPR system sufficient to forma CRISPR complex, including the guide sequence to be tested, may beprovided to a host cell having the corresponding target sequence, suchas by transfection with vectors encoding the components of the CRISPRsequence, followed by an assessment of preferential cleavage within thetarget sequence, such as by Surveyor assay as described herein.Similarly, cleavage of a target polynucleotide sequence may be evaluatedin a test tube by providing the target sequence, components of a CRISPRcomplex, including the guide sequence to be tested and a control guidesequence different from the test guide sequence, and comparing bindingor rate of cleavage at the target sequence between the test and controlguide sequence reactions.

CRISPR-Cas technology which facilitates genome engineering in a widerange of cell types is evolving rapidly. It has recently been shown thatdelivery of the Cas9-gRNA editing tools in the form ofribonucleoproteins (RNPs) yields several benefits compared with deliveryof plasmids encoding for Cas9 and gRNAs. Benefits include faster andmore efficient editing, fewer off-target effects, and less toxicity.RNPs have been delivered by lipofection and electroporation butlimitations that remain with these delivery methods, particularly forcertain clinically relevant cell types, include toxicity and lowefficiency. Accordingly, there is a need to provide a vector-free e.g.,viral vector-free, approach for delivering biologically relevantpayloads, e.g., RNPs, across a plasma membrane and into cells. “Cargo”or “payload” are terms used to describe a compound, or composition thatis delivered via an aqueous solution across a cell plasma membrane andinto the interior of a cell.

The current subject matter relates to delivery technology thatfacilitates delivery of a broad range of payloads to cells with lowtoxicity. Genome editing may be achieved by delivering RNPs to cellsusing some aspects of the current subject matter. Levels declinethereafter until Cas9 is no longer detectable. The delivery technologyper se does not deleteriously affect the viability or functionality ofJurkat and primary T cells. The current subject matter enables geneediting via Cas9 RNPs in clinically relevant cell types with minimaltoxicity.

The transient and direct delivery of CRISPR/Cas components such as Casand/or a gRNA has advantages compared to expression vector-mediateddelivery. For example, an amount of Cas, gRNA, or RNP can be added withmore precise timing and for a limited amount of time compared to the useof an expression vector. Components expressed from a vector may beproduced in various quantities and for variable amounts of time, makingit difficult to achieve consistent gene editing without off-targetedits. Additionally, pre-formed complexes of Cas and gRNAs (RNPs) cannotbe delivered with expression vectors.

In one aspect, the present subject matter describes cells attached to asolid support, (e.g., a strip, a polymer, a bead, or a nanoparticle).The support or scaffold may be a porous or non-porous solid support.Well-known supports or carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, gabbros, and magnetite. The natureof the carrier can be either soluble to some extent or insoluble for thepurposes of the present subject matter. The support material may havevirtually any possible structural configuration. Thus, the supportconfiguration may be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, or test strip,etc. Preferred supports include polystyrene beads.

In other aspects, the solid support comprises a polymer, to which cellsare chemically bound, immobilized, dispersed, or associated. A polymersupport may be a network of polymers, and may be prepared in bead form(e.g., by suspension polymerization). The cells on such a scaffold canbe sprayed with payload containing aqueous solution according to theinvention to deliver desired compounds to the cytoplasm of the scaffold.Exemplary scaffolds include stents and other implantable medical devicesor structures.

The present subject matter further relates to apparatus, systems,techniques and articles for delivery of payloads across a plasmamembrane. The present subject matter also relates to an apparatus fordelivering payloads such as proteins or protein complexes across aplasma membrane. The current subject matter may find utility in thefield of intra-cellular delivery, and has application in, for example,delivery of molecular biological and pharmacological therapeutic agentsto a target site, such as a cell, tissue, or organ.

In some implementations, an apparatus for delivering a payload across aplasma membrane can include an atomizer having at least one atomizeremitter and a support oriented relative to the atomizer. The methodfurther comprises the step of atomizing the payload prior to contactingthe plasma membrane with the payload.

The atomizer can be selected from a mechanical atomizer, an ultrasonicatomizer, an electrospray, a nebuliser, and a Venturi tube. The atomizercan be a commercially available atomizer The atomizer can be anintranasal mucosal atomization device. The atomizer can be an intranasalmucosal atomization device commercially available from LMA Teleflex ofNC, USA. The atomizer can be an intranasal mucosal atomization devicecommercially available from LMA Teleflex of NC, USA under cataloguenumber MAD300.

The atomizer can be adapted to provide a colloid suspension of particleshaving a diameter of 30-100 μm prior to contacting the plasma membranewith the payload. The atomizer can be adapted to provide a colloidsuspension of particles having a diameter of 30-80 μm. The atomizer canbe adapted to provide a colloid suspension of particles having adiameter of 50-80 μm.

The atomizer can include a gas reservoir. The atomizer can include a gasreservoir with the gas maintained under pressure. The gas can beselected from air, carbon dioxide, and helium. The gas reservoir caninclude a fixed pressure head generator. The gas reservoir can be influid communication with the atomizer emitter. The gas reservoir caninclude a gas guide, which can be in fluid communication with theatomizer emitter. The gas guide can be adapted to allow the passage ofgas therethrough. The gas guide can include a hollow body. The gas guidecan be a hollow body having open ends. The gas guide can include ahollow body having first and second open ends. The gas guide can be ahollow body having first and second opposing open ends. The diameter ofthe first open end can be different to the diameter of the second openend. The diameter of the first open end can be different to the diameterof the second open end. The diameter of the first open end can begreater than the diameter of the second open end. The first open end canbe in fluid communication with the gas reservoir. The second open endcan be in fluid communication with the atomizer emitter.

The apparatus can include a sample reservoir. The sample reservoir canbe in fluid communication with the atomizer The sample reservoir can bein fluid communication with the atomizer emitter. The gas reservoir andthe sample reservoir can both be in fluid communication with theatomizer emitter.

The apparatus can include a sample valve located between the samplereservoir and the gas reservoir. The apparatus can include a samplevalve located between the sample reservoir and the gas guide. The samplevalve can be adapted to adjust the sample flow from the samplereservoir. The sample valve can be adapted to allow continuous orsemi-continuous sample flow. The sample valve can be adapted to allowsemi-continuous sample flow. The sample valve can be adapted to allowsemi-continuous sample flow of a defined amount. The sample valve isadapted to allow semi-continuous sample flow of 0.5-100 μL. The samplevalve can be adapted to allow semi-continuous sample flow of 10 μL. Thesample valve can be adapted to allow semi-continuous sample flow of 1 μLto an area of 0.065-0.085 cm².

The atomizer and the support can be spaced apart. The support caninclude a solid support. The support can include a plate includingsample wells. The support can include a plate including sample wellsselected from 1, 6, 9, 12, 24, 48, 384, 1536 or more wells.Alternatively, the support comprises a plate, e.g., a scaled upconfiguration that can accommodate a monolayer with more cells than amicrotiter plate. The solid support can be formed from an inertmaterial. The solid support can be formed from a plastic material, or ametal or metal alloy, or a combination thereof. The support can includea heating element. The support can include a resistive element. Thesupport can be reciprocally mountable to the apparatus. The support canbe reciprocally movable relative to the apparatus. The support can bereciprocally movable relative to the atomizer. The support can bereciprocally movable relative to the atomizer emitter. The support caninclude a support actuator to reciprocally move the support relative tothe atomizer The support can include a support actuator to reciprocallymove the support relative to the atomizer emitter. The support caninclude a support actuator to reciprocally move the support relative tothe longitudinal axis of the atomizer emitter. The support can include asupport actuator to reciprocally move the support transverse to thelongitudinal axis of the atomizer emitter.

The longitudinal axis of the spray zone can be coaxial with thelongitudinal axis or center point of the support and/or the circularwell of the support, to which the payload is to be delivered. Thelongitudinal axis of the atomizer emitter can be coaxial with thelongitudinal axis or center point of the support and/or the circularwell of the support. The longitudinal axis of the atomizer emitter, thelongitudinal axis of the support, and the longitudinal axis of the sprayzone can be each coaxial. The longitudinal length of the spray zone maybe greater than the diameter (may be greater than double) of thecircular base of the spray zone (e.g., the area of cells to which thepayload is to be delivered).

The apparatus can include a valve located between the gas reservoir andthe atomizer The valve can be an electromagnetically operated valve. Thevalve can be a solenoid valve. The valve can be a pneumatic valve. Thevalve can be located at the gas guide. The valve can be adapted toadjust the gas flow within the gas guide. The valve can be adapted toallow continuous or semi-continuous gas flow. The valve can be adaptedto allow semi-continuous gas flow. The valve can be adapted to allowsemi-continuous gas flow of a defined time interval. The valve can beadapted to allow semi-continuous gas flow of a one second time interval.The apparatus can include at least one filter. The filter can include apore size of less than 10 μm. The filter can have a pore size of 10 μm.The filter can be located at the gas guide. The filter can be in fluidcommunication with the gas guide.

The apparatus can include at least one regulator. The regulator can bean electrical regulator. The regulator can be a mechanical regulator.The regulator can be located at the gas guide. The regulator can be influid communication with the gas guide. The regulator can be aregulating valve. The pressure within the gas guide can be 1.0-2.0 bar.The pressure within the gas guide can be 1.5 bar. The pressure withinthe gas guide can be 1.0-2.0 bar, and the distance between the atomizerand the support can be less than or equal to 31 mm. The pressure withinthe gas guide can be 1.5 bar, and the distance between the atomizer andthe support can be 31 mm The pressure within the gas guide can be 0.05bar per millimeter distance between the atomizer and the support. Theregulating valve can be adapted to adjust the pressure within the gasguide to 1.0-2.0 bar. The regulating valve cam be adapted to adjust thepressure within the gas guide to 1.5 bar. The or each regulating valvecan be adapted to maintain the pressure within the gas guide at 1.0-2.0bar. The or each regulating valve can be adapted to maintain thepressure within the gas guide at 1.5 bar.

The apparatus can include two regulators. The apparatus can includefirst and second regulators. The first and second regulator can belocated at the gas guide. The first and second regulator can be in fluidcommunication with the gas guide. The first regulator can be locatedbetween the gas reservoir and the filter. The first regulator can beadapted to adjust the pressure from the gas reservoir within the gasguide to 2.0 bar. The first regulator can be adapted to maintain thepressure within the gas guide at 2.0 bar. The second regulator can belocated between the filter and the valve.

The atomizer emitter can be adapted to provide a conical spray zone(e.g., a generally circular conical spray zone). The atomizer emittercan be adapted to provide a 30° conical spray zone. The apparatusfurther can include a microprocessor to control any or all parts of theapparatus. The microprocessor can be arranged to control any or all ofthe sample valve, the support actuator, the valve, and the regulator.The apparatus can include an atomizer having at least one atomizeremitter; and a support oriented relative to the atomizer; the atomizercan be selected from a mechanical atomizer, an ultrasonic atomizer, anelectrospray, a nebuliser, and a Venturi tube. The atomizer can beadapted to provide a colloid suspension of particles having a diameterof 30-100 μm. The apparatus can include a sample reservoir and a gasguide, and a sample valve located between the sample reservoir and thegas guide. The sample valve can be adapted to allow semi-continuoussample flow of 10-100 μL. The atomizer and the support can be spacedapart and define a generally conical spray zone there between; and thedistance between the atomizer and the support can be approximatelydouble the diameter of the circular base of the area of cells to whichmolecules are to be delivered; the distance between the atomizer and thesupport can be 31 mm and the diameter of the circular base of the areaof cells to which molecules are to be delivered can be 15.5 mm. Theapparatus can include a gas guide and the pressure within the gas guideis 1.0-2.0 bar. The apparatus can include at least one filter having apore size of less than 10 μm.

The aqueous solution and/or composition can be saponin-free.

The details of one or more variations of the subject matter describedherein are set forth in the accompanying drawings and the descriptionbelow. Other features and advantages of the subject matter describedherein will be apparent from the description and drawings, and from theclaims.

Optimisation of T Cell Preparation for Soluporation

Optimization of T cell culture conditions for maximal efficiency of mRNAdelivery using the vector-free reversible permeabilization method isdescribed below. The vector-free method for intracellular delivery ofmacromolecules and nucleic acids described herein has demonstratedsuccess in facilitating the delivery of gene-editing tools such asCRISPR/Cas9 and mRNA to mammalian cells such as primary human immunecells. Culture conditions for efficient transfer of mRNA to human Tcells using the vector-free delivery platform were determined.

Considerable differences exist in the ex vivo activation of humanlymphocytes amongst clinical groups and T-cell engineering companies.There is no single standardised protocol for expansion of primary humanT cells, therefore, a comprehensive evaluation of culture media,additives, activation methods and timing schedules was undertaken toidentify the optimal ex vivo culturing conditions that promote maximalefficiency transfer of mRNA using the vector-free reversiblepermeabilization method technology. Herein, the cell isolation protocoland the results/conclusions of each experiment undertaken is described.

To address suspension cells (i.e., non-adherent cells), a centrifugationstep was developed as part of the delivery process. This step wasdeveloped to enable formation of an exposed monolayer of suspensioncells. The centrifugation step allows for both formation of themonolayer and removal of the supernatant in one step. In contrast, withadherent cells, the cells were already in monolayer formation and mediumwere removed by pipetting.

Materials and Methods

Cell culture and transfection. Human peripheral blood mononuclear cells(PBMC) were recovered by centrifugation over a Percoll gradient fromLeuko Pak (AllCells Alameda, CA). CD3⁺ enriched lymphocytes wereisolated by magnetically activated cell sorting using CD3 Microbeads(Miltenyi). Cells were cryopreserved in 10% dimethyl sulphoxide (DMSO)and foetal bovine serum (FBS). Following initial thawing from stockaliquots, CD3⁺ T cells were cultured in human recombinant interleukin-2(IL-2) with primary and co-stimulatory antibody activation using variousprotocols (see below) in a humidified tissue culture incubator at 37° C.and 5% CO_(2.)

Delivery procedure. Activated T cells were seeded at 1.5×10⁶ cells perwell of a 96-well filter plate (Acroprep, 1.2 μm Supor membrane; Pall,USA). Media was removed from the wells by centrifugation at 300×g for 5min. 7 μl of delivery solution (32 mM sucrose, 12 mM potassium chloride,12 mM ammonium acetate, 5 mM HEPES and 27% ethanol in molecular gradewater (all from Sigma-Aldrich)) containing 4 μg GFP mRNA was thensprayed into each well using the vector-free delivery spray instrument.The atomizer used in the instrument was a MAD NasalTM intranasal mucosalatomization device (Wolfe Tory Medical Inc, Salt Lake City, USA). Theatomizer was held on a retort stand at 26 mm above the bottom of thewell and was connected to a 6 bar compressor (Circuit Imprimé Français,Bagneux Cedex, France) via polyurethane tubing (6 mm outside diameter, 4mm inside diameter; SMC, Tokyo, Japan). The delivery solution containingthe cargo was pipetted into a delivery port located at the top of theatomizer and the spray was generated at 1.5 Bar using a spray actuatorbutton (SMC, Tokyo, Japan). Following delivery, the cells were incubatedin this solution for 2 min prior to the addition of 50 μl Stop Solution(0.5× PBS). Thirty seconds later T cell media was added (100 μl) andcells were allowed to recover at 37° C. and 5% CO₂ overnight. Uptake andviability were assessed at 24 h post-delivery.

Cell viability, FACS Sample Preparation and Analysis. To assess cellviability following the vector-free method of delivery,7-Aminoactinomycin D (7-AAD) (Sigma) was used to stain the cells.Briefly, cells were in washed in PBS+1% foetal bovine serum (FACSbuffer) followed by incubation with 7-AAD (1:40 for 5-10 min protectedfrom light at room temperature), followed by resuspension in PBS+1% FBS(FACS buffer). Samples were processed on the BD Accuri C6 flow cytometer(Becton Dickinson, USA) and data was analysed using the C6 software.Cell debris was excluded from whole cells using forward and side scatterparameters. Single cells were selected by excluding doublets in the FSCheight vs FSC are plot. GFP expression was analysed on gated viablecells.

Media, Activation Reagents and Timing

Four media for T cell culture and expansion were evaluated using mRNA,e.g., model cargo GFP mRNA, as cargo and GFP expression as an indicatorof successful culture methodology. cRPMI was tested which is aserum-containing media typically used in the culture of primary immunecells. However, as serum is a highly variable supplement in cell-culturemedia, three serum-free and xeno-free expansion media, optimised for thein vitro culture of human T cells were also evaluated.

cRPMI was prepared using RPMI, heat-inactivated fetal bovine serum (PBS)(10% v/v), penicillin-streptomycin, L-glutamine and supplemented withIL-2 (100 U/ml). cRPMI was utilised as culture medium in experimentsthat assessed the performance of various T-cell proliferation protocols.The first proliferation protocol tested was ImmunoCult™ Human CD3/CD28T-cell Activator which consists of a soluble tetrameric antibody complexthat bind CD3 and CD28 cell surface ligands on the T lymphocytes. It wasevaluated alongside an alternative method of activation which use“feeder” cells as a means of presenting antigen to the T cell receptor(TCR) to induce proliferation of the T cell (FIG. 1).

T Cell Activation Using PBMC or A549 as Feeder Cells

Autologous PBMC were transferred to the tissue culture plate. After 2 hrincubation, the supernatant was removed leaving the adhered monocytes. Tcells cultured in cRPMI were added to the plate and allowed toco-incubate with the monocytes for several days. In a similar protocol,the A549 cell line was left to adhere for up to 2 h. Medium was removedand T cells were added to the plate and left to co-incubate prior todelivery of mRNA using the vector-free delivery technology. Uptakeefficiencies using feeder cells were variable both inter andintra-experiment (FIG. 1), therefore, ImmunoCult™ Human CD3/CD28 T-cellActivator reagent was utilised in further evaluations.

T Cell Activation Using Dynabeads®

T cells are activated using methods known in the art, e.g., antibodiesthat bind to cell surface proteins such as CD3 and/or CD28, feedercells, and/or magnetic beads comprising immunostimulatory molecules. Forexample, Dynabeads® were examined as an alternative to ImmunoCult™ HumanCD3/CD28 T-cell Activator. Dynabeads® are superparamagnetic beads coatedwith antibodies against human CD3 and CD28 that provide the primary andco-stimulatory signals necessary for T cell activation and expansion.The bead-to-cell ratio recommended is 1:1, however, another conditionusing 3 beads per cell was also used to assess whether more efficientand rapid expansion could positively affect uptake of mRNA into T cellsusing the Soluporation delivery method. Increasing the bead-to-cellratio significantly improved percentage uptake (FIG. 2A). A repeatexperiment was then carried out to include the ImmunoCult™ HumanCD3/CD28 T-cell Activator at 3 times the concentration recommended bythe manufacturer, however, this concentration did not result in the sameimprovement in efficiency observed using Dynabeads (FIG. 2B)

Culture Medium: Prime XV

Cell-compatible culture media are used in the delivery methods. Forexample, Prime-XV (Irvine Scientific) is a first serum-free and animalcomponent free media that was tested with vector-free deliverytechnology. Based on positive data observed using Dynabeads at a 3:1bead to cell ratio (FIG. 2 and FIGS. 2A and 2B), it was decided tocontinue using this method of activation to test alternative culturemedia. This medium was tested using Dynabeads to induce proliferationalongside an alternative activation method which stimulates T cellproliferation by binding anti-human CD3 antibody to cell culture platesfollowed by the addition of soluble anti-CD28 to the media.Dynabead-activated T cells cultured in Prime XV demonstratedsignificantly better uptake efficiency (up to 50%) compared to thosecells stimulated using soluble a-CD3/CD28 (<20%) (FIG. 3).

Although delivery to T cells cultured in Prime XV did improve uptake ofmRNA using the vector-free technology, it was associated withcell-handling issues, e.g., removing the cells from culture 24 h afterinitial seeding and recovery of cells from the Dynabeads upon washing(FIGS. 4A and 4B).

Culture Medium: Supplementary Cytokines

In some examples, the cell culture media was supplemented with a higherconcentration of IL-2 (200 U/ml instead of 100 U/ml) to enhance the rateof proliferation (Tumeh P, et al., J Immunother 2010. 33(6): 759-768 andBesser M J, et al., Cytotherapy 2009. 11:206-217).

Culture Medium: Immunocult

Immunocult™-XF Expansion Medium (StemCell Technologies) was evaluated.Like Prime XV, it is also a serum-free, xeno-free T cell culture medium.In this example, T cells were cultured in Immunocult Expansion Mediumand activated using Dynabeads® bead to cell ratio of 3 to 1. GFP mRNAwas delivered to the cells at day 1 and day 2 post-activation andassessed for uptake 24 h later (FIG. 5).

Timing of Delivery Post-Activation of T Cells

Cells are activated for between 15 to 21 hr, with 19 hr being preferred.

A preferred “window” for delivery post-activation after addition ofDynabeads was identified. mRNA was delivered using the vector-freetechnology at several time points. Optimal GFP expression was observedwhen mRNA payload, e.g., model payload GFP mRNA, was delivered at 19 hpost-activation compared with 17 h and 21 h (FIG. 6). Studies wereundertaken to determine if there was a correlation between increasedcell size and time post-activation. Cell size was estimated using theforward scatter (FSC) data obtained from flow cytometry analysis. It wasobserved that maximal transfection efficiency correlated with a timewhen cells were actively increasing in size. Exemplary results aredepicted in Table below:

TABLE Timing of delivery post-activation of T cells Time post-activationwhen GFP mRNA delivered 17 h 19 h 21 h GFP expression at 24 hpost-delivery 16.78% 24.23% 16.77% (Mean of 4 replicates) Size (FSC)2378956 2449816 2514971

T Cell Activation Using TransAct

T cell TransAct™ (Miltenyi) is a colloidal reagent consisting of ananomatrix conjugated to CD3 and CD28 agonist which provide signals foractivation and expansion of T cells. It provides benefits overDynabeads® as excess reagent can be removed by centrifugation withoutthe need for magnetic separation and the beads from the cells and allthe subsequent washing. Thus, this reagent was evaluated over athree-day period using Immunocult as the culture medium to determine thepreferred day for delivery of mRNA by the vector-free deliverytechnology described herein. TransAct initiates T cell proliferationless aggressively than Dynabeads. Therefore, optimum delivery of mRNA toT cells was observed 24 h later than that demonstrated using beadactivation, however, this was accompanied by an enhancement intransfection efficiency (FIG. 7).

Culture Medium: TexMACS

TexMACS (Miltenyi Biotech) was also tested as an alternative serum-freemedium for T-cell culture. This T cell stimulation and expansion reagentwas useful but not routinely used going forward (FIG. 8).

Effect of Post-Thaw Recovery Period Prior to Activation

The benefit for delivery to allow cells to recover overnight beforeaddition of activation reagent compared with adding the recovery agentimmediately after thawing without a recovery period was evaluated. Uponthawing of T cells from liquid nitrogen storage, cells were left torecover overnight in culture medium alone before the addition ofactivation reagent. This step resulted in a 30% improvement intransfection efficiency. In some examples, fresh primary non-adherentcells are used in the method; in other examples, primary non-adherentcells are frozen, e.g., for storage, and then thawed prior to thepayload deliver method. The method may thus optionally include afreeze/thaw step of primary non-adherent cells. These data indicate thata recovery period (after thawing) prior to activation is useful.

Cell Culture Density

Cells were cultured in Immunocult Expansion medium at various seedingdensities (1×10⁶/ml and 5×10⁶ /ml) prior to the vector-free delivery.The density at which cells are cultured prior to Soluporation is between1-5×10⁶/mL]. Higher seed number led to an enhancement in uptakeefficiency (FIG. 10).

T Cell Activation Using Zinc

Zinc influx can support T cell activation (Yu M, et al., J. Exp. Med.208 (4) :775-785) but may also improve transfection of nucleic acid(Niedzinski E J, et al. Mol Ther 2003 7(3): 396-400). Zinc improved Tcell proliferation in two independent experiments (FIG. 11). A trendtowards an enhancement of transfection efficiency was observerd. Thus,zinc is an optional component of the cell culture medium at theactivation step The range of zinc concentration ranges from 0.03 mM to 3mM.

Optimization of T Cell Culture Conditions for Maximal Efficiency of mRNADelivery

Evaluation of multiple culture media, activation methods,supplementation and time courses has resulted in a preconditioningprotocol that maximises the transfection efficiency of mRNA, e.g., themodel payload GFP mRNA, to human primary T cells using the vector-freedelivery technology. In this evaluation, cells were cultured inImmunocult™ T Cell Expansion Medium supplemented with 200 U/ml of IL-2at a density of 5×10⁶/ml. Cells were left to recover overnight post-thawbefore the addition of 1× T cell TransAct™ (Miltenyi) for 48 h prior tovector-free delivery of nucleic acid.

Human peripheral blood mononuclear cells (PBMC) were recovered bycentrifugation over a Percoll gradient from Leuko Pak (AllCells Alameda,Calif.). CD4 enriched T cells were isolated by negative selection torecover a purified population using anti-CD8 microbeads and theflow-through was collected from an LD column (Miltenyi). Cells werecultured in standard cell culture media, e.g., complete RPMI using RPMIbasal medium, heat-inactivated fetal bovine serum (FBS) (10% v/v),penicillin-streptomycin, L-glutamine and supplemented with IL-2 (200U/ml). Cells were left to recover for 4 hours before the additionDynabeads® at a bead to cell ratio of 3 to 1. mRNA was delivered to thecells at day 1 post-activation and assessed for uptake 24 h later.Multiple hits in this cell type achieved higher uptake efficiency, >30%(FIG. 12).

T cells were enriched from PBMC cultured in X-VIVO 15 supplemented with2 mM GlutaMAX, 10 mM HEPES and 5% Human AB Serum and 250 IU/ml IL-2.Cells were seeded at a density of 1×10⁶/mL and supplemented withanti-CD3 and anti-CD28 antibodies (Miltenyi) prior to culture. mRNA,e.g. test payload GFP mRNA, was delivered to the cells by soluporationat either day 2 or day 3 post-initiation and assessed for uptake 24 hlater (FIG. 13).

Optimisation of T Cell Monolayer Formation

A cell monolayer is a culture in which cells are oriented in a singlelayer on substrate. The substrate is generally a plate, e.g., amicrotiter plate, a flask, Petri dish, membrane, or filter upon whichthe cells lie. In cell culture, a monolayer refers to a layer of cellsin which cells are substantially side by side and often touching eachother on the same surface. The cells are adherent cells (cells thatattach to a substrate) or non-adherent cells (cells that float or aresuspended in culture media). Adherent cells grow on a substrate, attach,and thereby form a monolayer. A monolayer can also be made fromnon-adherent or “suspension cells”. The terms “non-adherent cells” and“suspension cells” are used interchangeably herein.

A number of techniques may be employed to make a cell monolayer fromnon-adherent or suspension cells prior to delivery of a payload to thecells. Such techniques include allowing a culture suspension cells tosettle on substrate, centrifugation, exposure to a vacuum, exposure topositive pressure, use of magnetic T -cell activation beads and/ordeposition onto a membrane (e.g., the use of a transwell insert system,described below.)

Transwell Insert

In order to form a monolayer of suspension cells that would allow thecells to be presented optimally to the spray, a transwell insert systemwas used. Cells were seeded at 1×10⁶ in 400 μl per insert and the mediawas removed by placing the transwell insert (Greiner bio-one; CAT#655640; 12 Well ThinCert; PET 0.4 μm) into a device that allowed avacuum to be applied to the bottom of the insert (between −0.5 bar and−0.65 bar; see FIGS. 14A, B, C). Once the media was removed theremaining cells formed a monolayer to which the spray can be applied.Non-adherent cells such as PBMCs, primary T-cells, or a cell line, e.g.,Jurkat T cells, were added to the insert system, the vacuum applied andthe insert placed into a 12 well plate and sprayed with deliverysolution (10 μl) containing test payload such as eitherfluorescently-labelled beta-lactoglobulin (BLG), bovine serum albumin(BSA) or ovalbumin (OVA). Stop solution (50 μl) was applied after a 2minute incubation and 30 s later normal media (100 μl) was applied.Expression levels of 55.6, 28.5 and 15.3% were achieved (FIG. 15). Theinsert system was found to be useful as an exemplary technique togenerate a monolayer using non-adherent cells.

96-Well Polyethersulfone (PES) Plate

Permeable membranes that allow filtering of culture media are alsouseful to generate cell monolayers. Such membranes include cellulosenitrate membranes, cellulose acetate or PES membranes.

Such a membrane-based system was assessed to create a suspension cellmonolayer. 96-well filter bottomed plates provide a 96-well format witha filter bottom to the well. The Pall Supor filter plate (AcroPrepAdvance; PES 1.2 pm, CAT #8039) was assessed. 1×10⁶ human primary Tcells were seeded in 100 μl per well and the plate centrifuged at 300×gfor 5 min. Once the media was removed by centrifugation, and theremaining cells formed a monolayer, the plate was placed within theSolupore device and cells sprayed with delivery solution containingmRNA. Stop solution (50 μl) was applied after a 2 minute incubation and30 s later normal media (100 μl) was applied. The cells were incubatedfor 2 hours before the process was repeated. At the end of this spraythe cells were incubated overnight at 37° C. and 5% CO₂ in a humidifiedincubator and assessed for GFP fluorescence by flow cytometry. GFPexpression levels of 52%±3.6 across 5 donors and 5 experiments wasachieved with a viability of 97%±3.3 (FIGS. 16A, B, C). The PES platecontains a mesh-like filter in which cells may become irretrievable.Other filter types, such as track-etched, were assessed. A track-etchedmembrane is a thin (˜5-25 microns) polymer membrane the pores of whichare formed by irradiating the initial non-porous material withhigh-energy particles and subsequent etching (usually by caustic etchant(for example NaOH)) of latent tracks to form pores through the membraneof a given diameter.

96-Well Polycarbonate Track-Etched (PCTE) Plate

Alternative membrane filter systems may be used for generation of a cellmonolayer of non-adherent cells. For example, an alternative filterplate with 0.4 μm hydrophilic PCTE filter was obtained from Agilenttechnologies. Human primary T cells were seeded at 2.5×10⁵ cells per 100μl per well and centrifuged at 350×g for 2 min Once the media wasremoved and the cell monolayer formed, the plate was placed within theSolupore device and cells sprayed with delivery solution containing atest payload such as GFP mRNA. Stop solution (50 μl) was applied after a2 minute incubation and 30 s later normal media (100 μl) was applied.The cells were incubated for 2 hours before the process was repeated. Atthe end of this spray the cells were incubated overnight at 37° C. and5% CO₂ in a humidified incubator and assessed for GFP fluorescence byflow cytometry. GFP expression levels of 72%±5 was achieved with aviability of 75.0%±3.5 (FIG. 17). The results of a comparison of PES andPCTE plates is shown in FIG. 18 and shows that uptake is enhanced whenusing the PCTE plates. Thus, a hydrophilic membrane filter, optionallytrack-etched, is a useful exemplary membrane and is preferred in someembodiments.

Media Removal

The method for removal of media from the cells was also addressed. Usingthe filter plates, centrifugation, vacuum pressure and positive pressurewere assessed. 1×10⁶ human primary T cells were seeded per well in a96-well filter plate (Pall; Supor, 1.2 μm; CAT #8039). The media wasremoved by either centrifugation at 300×g for 5 min or by vacuumpressure (−20 mBar, 30 s; see FIG. 105-107). The cell monolayer wassprayed with 4 μl of delivery solution containing 0.57 μg/μl of GFPmRNA. Stop solution (50 μl) was applied after a 2 minute incubation and30 s later normal media (100 μl) was applied. The cells were incubatedovernight at 37° C. and 5% CO₂ in a humidified incubator and assessedfor GFP fluorescence by flow cytometry (FIG. 19A, B). In anotherexperiment, human primary T cells were seeded at 2.5×10⁵ cells in 100 μlper well of the Agilent PCTE filter plate (Agilent; PCTE 0.4 μm). Themedia was removed by either centrifugation (350×g for 2 min), orpositive pressure (200 mBar for 1 min;). Once the media was removed andthe cell monolayer formed the plate was placed within the Soluporedevice and cells sprayed with delivery solution containing GFP mRNA.Stop solution (50 μl) was applied after a 2 minute incubation and 30 slater normal media (100 μl) was applied. The cells were incubated for 2hours before the process was repeated. At the end of this spray thecells were incubated overnight at 37° C. and 5% CO₂ in a humidifiedincubator and assessed for GFP fluorescence by flow cytometry (FIG. 19A,B). Vacuum pressure is used to remove the media from the wells toprovide a cell monolayer. Optionally, both positive pressure andcentrifugation are used to produce monolayers. In some embodiments, thelatter technique is preferred.

Magnetic Beads

An alternative method combines the use of the T cell activation beads(e.g., DynaBeads 3:1 ratio) and a magnet. After overnight activation,the T cells (bound to the DynaBeads) were seeded in a 96-well plate. Amagnet was placed underneath the wells and the media removed by pipette.The magnet holds the beads and cells in place while the media isremoved. mRNA was then delivered by Soluporation. GFP expression wasdetected 24 hr later by fluorescence microscopy.

mRNA Delivery to MSCs

To confirm delivery to cells in a monolayer, mesenchymal stromal cells(either primary human or iPSC-derived) were seeded in 96-well plates sothat by 24 hrs the confluency was 80-90%.

Delivery of mRNA, e.g., test payload/cargo GFP mRNA, to BM-MSCs andiPSC-MSCs was evaluated. The delivery of various cargo compounds such as10 kDa dextran to primary human BM-MSCs using a vector-free method forintracellular delivery involving reversible permeabilization waspreviously reported (O'Dea S, et al., PLoS One. 2017.30;12(3):e0174779). A method for delivery of functional molecules suchas mRNA was also evaluated. A reporter GFP mRNA was used to evaluatemRNA delivery efficiency to BM-derived and iPSC-derived MSCs.

Multiple treatments, e.g., three doses of GFP mRNA, were delivered toBM-MSCs and iPSC-MSCs over 2 days. Fluorescence microscopy confirmedexpression of GFP protein in cells 24 hr following delivery of the finaldose of mRNA (FIG. 72A). Flow cytometry analysis indicated deliveryefficiencies of 29.5±10.4% and 31.0±2.2% (n=3) in BM-MSCs and iPSC-MSCsrespectively (FIG. 21A, B).

Optimisation of Atomisation of Delivery Solution

The Mucosal Atomization Device (MAD Nasal™) spray head, used to atomizethe payload solution dispenses volume of milliliters while soluporationworks with micro-litres volumes. The use of a microliter volumeatomizer/droplet delivery system is preferable.

Alternative spray heads were investigated for two main purposes:increasing the uptake and improving reproducibility across replicates(intra-experiment) and across experiments (inter-experiment). Anassessment of alternative atomiser devices was undertaken to find whichwas optimal for mRNA delivery to T cells. The atomiser allows theapplication of the delivery solution in dropletised form to the cellmonolayer. In addition to identification of the atomiser, a controllerdevice was designed and built which allowed fine control of theatomisation process. A variety of parameter sets were tested to find theoptimal parameters for delivery of mRNA to T-cells.

The results indicated that a microliter volume delivery device such asthe Ari Mist nebuliser and 180 kHz ultrasonic nebuliser gave comparableuptake and reproducibility while cell viability was slightly higher withAri Mist. Such a microliter volume delivery device such as the Ari Misthead was preferred and can be incorporated into an automated solutionsince it is smaller in size, easier to handle and does not require apower box to operate. Other units such as the Burgener nebulisationtechnology (U.S. Pat. No. 6,634,572, hereby incorporated by reference)also was suitable for the scaling up of soluporation. Taken together,the Ari Mist and other Burgener nebulizers were used moving forward forautomation and scaling.

Atomization of the Delivery Solution to Produce Monodispersed Droplets

A cell membrane permeabilizing solution can be delivered onto amonolayer of cells using a variety of methods. For example, thepermeabilizing solution can be atomized using ultrasonication or it canbe nebulized using a pneumatic nebulizer.

Both air-assisted ultrasonication and pneumatic nebulization were testedas delivery methods. A total of eight different spray heads were tested:three ultrasonic heads namely 60 KHz (Sonaer), 130 kHz (Sonaer) and 180kHz (Sonotek Echo) and five pneumatic nebulizers namely Ari Mist, X-175,PFA 250, T2100 and Peek Mira Mist (Burgener Research)

Ultrasonication tests were performed at 60 kHz, 130 kHz, and 180 kHz.Liquid can be driven to an ultrasonic nozzle by a pumping system, and itcan be atomized into a fine mist spray using high frequency soundvibrations.

A curtain of gas (air) can assist the process. An auxiliary piece calledshaper is mounted around the ultrasonic head and the function of the airis to shape the mist of liquid ultrasonicated. For example, the air hadthe dual function of shaping the spray as well as promoting the payloadto enter the cells. This was possible because the air fed through theshaper was at pressure beyond that used for shaping function.

Testing with the ultrasonic nebulizer yielded results useful fordelivery of caro/payload to mammalian cells, e.g., non-adherent cells.Up to 60% Dextran-Alexa488 (model cargo) was delivered to U2OS cellsusing the 180 kHz ultrasonic head (FIG. 22A, B).

Piezoelectric transducers can be used to impart electrical input intomechanical energy in the form of vibrations, which created capillarywaves in the liquid when introduced into the nozzle, and resulted inatomization of the liquid. Each ultrasonic probe worked at a givenresonant frequency. The operating frequency can determine the size ofthe liquid droplets generated. The size of the droplets can also beaffected to a lesser extent by the power at which the ultrasonic probeis operated. An ancillary air stream can be used to help control andshape the spray.

An exemplary ultrasonic spray emitter generates a fine spray, withnarrow size distribution of the droplets which in turn results in evendeposition of the delivery solution and payload onto cells. Reducing thevolume delivered and reducing the ethanol concentration, e.g., withrespect to the preferred parameters evaluated for the MAD nasal sprayhead, improved delivery efficiency and improved viability with theultrasonic spray head.

Additional nebulizers to generate droplets (microliter volume) weretested such as Ari Mist, Peek Mira Mist, T2100, X175 and PFA250nebulizer (Burgener Research). These exemplary nebulizers operate oncompressed gases and require a pump to supply the sample solution. Theseexemplary atomizers have two parallel channels, one for the gas (air)and the other one for the liquid to be nebulized. Both paths end at thetip of the nebulizer with an orifice for the gas and an exit for theliquid. The gas flow can draw the liquid into the gas stream. The impactwith the gas molecules can break the liquid into small droplets,resulting in nebulization.

The Burgener nebulizers (U.S. Pat. No. 6,634,572) tested differ in innerdiameter, material and optimal flow rate. In preliminary tests, thenebulizers gave comparable expression of cargo mRNA. Characterisation ofthe droplet size revealed droplets ranging from 1-20 μm, with the peaknumber of droplets in the range of 5-7 μm. The average particle size asdefined by the Sauter mean (D₃₂) dimeter is 13 μm (seehttp://www.burgener.com/EnhancedData.html)] Amongst the suite ofBurgener's nebulizers tested the Ari Mist was chosen as the preferentialspray head based on several factors: uptake and viability were good, itsspecs (optimal flow-rate, inner diameter) matched the characteristics ofthe pumping system, its inner diameter (225 μm) was small enough tohandle low volumes of liquid without the inconvenience of clogging.

Certus Digital Dispensing Technology

The Certus Flex liquid dispensing instrument was equipped with an 8channel dispensing head (Cat. #D196057) and two valve sizes (0.10 nozzlediameter, 0.03 travel (Cat. #21765) and 0.15 nozzle diameter, 0.03travel (Cat. #21766). Each channel is individually controlled using theCertus proprietary software and electronics. The Certus Flex enablescontactless dispensing of liquid and large molecules using Gyger microvalve technology and air pressure control. Volumes in the nano litre(nl) range can be delivered with high precision (100 nl with CV 5%; CVrepresents coefficient of variation or relative standard deviation).

Delivery mRNA to T cells through the generation of small droplets in thenanoliter (nl) to μl size range was examined to evaluate the feasibilityof delivering mRNA to T-cells using the Certus Flex microfluidicplatform.

CD3⁺ T-cells were activated using, e.g., either Dynabeads or TransAct.Cells were seeded at 1.5×10⁶ cells per well of a 96-well filter plate(PES). The plate was centrifuged, e.g., for 5 mins at 300×g to removethe cell culture medium. Delivery solution was dispensed to each wellthrough Channel 1 with the parameters listed in Table 1. A total volumeof microliter amounts, e.g., 2 or 7 μl, was delivered in dropletsranging in volume from 7 to 0.08 μl. The volume of the droplet wasdetermined by the number of drops dispensed into the well. FIG. 58,represents the droplet array pattern tested (FIG. 58). The valve type,pressure and height were varied as outlined in Table 2. Cells wereincubated for 2 minutes following application of the delivery solution.50 μl Stop solution was added though Channel 2 and incubated for 30 s.100 culture medium was added though Channel 3. The plates were incubatedat 37 degrees for 24 hrs prior to analysis (FIG. 58).

The results indicate that cell viability was comparable to untreatedcells using this system. No delivery of GFP mRNA was observed using thissystem. This was seen across all parameters tested (FIG. 61). Thus,using the Certus Digital Dispensing Technology to delivery droplets inthe nl-μl range did not result in uptake of GFP mRNA to T-cells.

TABLE 1 Plate Delivery Template Parameters Volume per Volume per PointsPoints point (2 μl point (7 μl Orientation per plate per well delivery)delivery) 1 × 1 96 1 2 7 2 × 2 1761 4 0.5 1.75 3 × 3 4001 9 0.2222222220.777777778 4 × 4 6901 16 0.125 0.437 5 × 5 11021 25 0.08 0.28

TABLE 2 Dispensing head channel configuration Dispense IncubationChannel Valve Solution Volume Pressure Height time 1 0.10, 0.03Delivery-  2/7 μl 0.3/0.6 15.5/31 mm 120 secs EGFP Bar mRNA 2 0.15, 0.03Stop  50 μl 0.3 Bar 15.5/31 mm  30 secs Solution 3 0.15, 0.03 Culture100 μl 0.3 Bar 15.5/31 mm  24 hrs (incubator) Medium

Instrumentation to Enable Fine Control of the Spray

A test rig was built to control the critical spray parameters and enablemechanisation of the spray. The plate containing the cell suspension wascentrifuged prior to being placed on the test rig. The delivery solutioncontaining the payload was loaded into the elveflow fluidic reservoir ora syringe system. Fluidic control of the delivery solution containingthe payload was brought about either using a pinch valve or using amicro valve. Addition of the stop and culture medium was done manually.

Fluidic control of the delivery solution containing the payload wasbrought about using two systems, an elveflow-pinch valve system and asyringe-micro valve system. The syringe-micro valve system was shown tohave benefit over the elveflow-pinch valve system.

a) Fluidic Control of the Delivery Solution Containing the Payload

(i) Elveflow-Pinch Valve

Elveflow refers to a microfluidic reservoir which was used with a 1.5 mlEppendorf tube or 50 ml falcon tube depending on the sample reservoirsize required (Elvesys, Innovation centre, 83 avenue Philippe Auguste,75011, Paris, FRANCE). Pinch valve can refer to any pinch valve where anexample is the Electronic Clippard pinch valve (Clippard, 7390 ColerainAvenue, Cincinnati, Ohio 45239, USA) The fluidic control can be achievedby fluid control system that can apply a constant pressure to anelveflow fluidic reservoir to drive the fluid through a pinch valve(FIG. 23). A volume of fluid that can be dispensed can be controlled by:an amount of pressure applied; a length of time the valve is open;and/or a diameter of the tubing used.

The valve can be activated by a metal-oxide-semiconductor field-effecttransistor (MOS FET) which can be controlled by a microprocessor.

(ii) Syringe-Micro Valve

-   -   The Elveflow-pinch valve system described above had limitations:        -   Calibration of the system did not hold when the elveflow            sample reservoir was reloaded.        -   There was poor accuracy and precision in dispensing volumes            lower than 5 μl (For low volumes (<5 μl) the relative            standard deviation was approximately 9% over repeated            dispenses These data were generated in Avectas and are            summarised in FIG. 54. Calibration data for the delivery            solution using the elveflow-pinch valve system.    -   To address these limitations, a new fluidic system was used.        This can involve using a micro valve fluidic system such as the        Gyger microvalve (SMLD300, Fritz Gyger AG, Bodmerstrasse 12,        3645 Gwatt (Thun), Switzerland). This system includes a syringe        sample reservoir connected to a micro valve which is connected        to the Air Mist nebulizer. This system had greater accuracy and        precision when delivering volumes in the range of 1 μl to 100        μl. A comparison of delivery efficiency using the microvalve and        pinch valve demonstrates no difference in delivery efficiency        (FIG. 63A, B)

b) Fluidic Control of the Air

-   -   Air pressure is optionally controlled by solenoid valve.

c) Electronics to Control Spray Actuation

-   -   To enable electronically controlled spray actuation, a system        was designed using a microprocessor based development board to        allow easy development of time controlled sequences. The        development board used the microprocessor, e.g., PIC16F1619. The        spray actuation time and fluid delivery time can be manipulated        through the development board's interface software. The        microprocessor development board enables pulsing of the        nebulizer spray.    -   This system was then upgraded to utilise the high speed and        repeatable PLC technology (programmable logic controller) to        better align with industry standards and to serve as proof of        concept for the automated Solupore™ technology (which is based        on ultra-high-speed Programmable Logic Controller (PLC)        technology). The Test Rig controller consisted of a PLC with a        Gyger controller and a program which communicates between the        two pieces of hardware. There is operator interaction to the        hardware via a momentary push button.

d) Alignment of the Spray Head

-   -   A sprayhead such as the Ari Mist nebulizer parallel path design        produces a spray which is off centre from the nebulizer tip.        Using a custom spray head holder equipped with a goniometer, the        alignment of the spray head can be adjusted.        Identification of Optimal Parameters for Delivery of mRNA into        T-Cells

Work was carried out to characterise and optimise the spray with thethree ultrasonic heads and Ari Mist head. The character of the varioussprays was assessed using high speed camera recording. The force of thespray experienced by the cell monolayer was determined by force sensoranalysis. In some cases, the volume delivered into the wells of a 96well plate was assessed using a colorimetric assay

For the optimisation study the following parameters were tested, in theranges indicated:

-   -   a) Air pressure: 0.5-2 bar    -   b) Volume delivered: 1-7 μl    -   c) Height of atomiser to target area: 26 and 31 mm    -   d) Length of spray actuation: 50-900 ms    -   e) Flow rate: 1-20 μl/s    -   f) Power of ultrasonic probe: 40-80%    -   g) Spray head: Ari Mist, Ultrasonic 60 kHz, Ultrasonic 130 kHz,        Ultrasonic 180 kHz    -   Numerous sets of parameter combinations were tested with the        three ultrasonic probes as well as with the Ari Mist nebulisers,        using EGFP mRNA as the payload.

All sets of parameters resulted in GFP expression, with uptake varyingfrom 5% to 30%. Amongst the ultrasonic emitters, 180 kHz proved to bemore effective compared to the 130 kHz and 60 kHz ultrasonic heads indelivering payloads to T-cells. The test results indicated that payloadshad been delivered to T-cells successfully, at an efficiency ofapproximately 15-28%, with high level of consistency between replicates(±1%). The health of the cells was maintained following delivery (85%relative viability).

Uptake and reproducibility obtained with Ari Mist, 180 kHz ultrasonicand Mad Nasal spray heads was compared. Results are presented in thelongitudinal data plots for Ari Mist, 180 kHz ultrasonic and Mad Nasalspray heads (FIGS. 24, 25, and 26, respectively). The longitudinal dataalso show the progress in the optimisation of the delivery parametersover the weeks. The uptake improved reaching 20-30% positive cells with180 kHz ultrasonic (FIG. 25) and Ari Mist (FIG. 24) while it fluctuatedbelow 20% when cells were soluporated using MAD nasal spray head (FIG.26).

Improved reproducibility was observed when ultrasonic or Burgenernebulisers were used (FIGS. 24, 25), in comparison to the MAD nasalspray head (FIG. 26). The reproducibility of the uptake was expressedthrough standard deviation (StDev). A major goal of evaluating otherspray heads was to identify nebulisers that gave standard deviation ofthe uptake within replicates of one experiment as narrow as possible.Considering the longitudinal data, by averaging the standard deviationof the uptake across all experiments, the 180 kHz ultrasonic spray headthe average standard deviation of the uptake was 2.4%, with Ari Mist was2.1% while with MAD nasal was 4.5%. In both cases there was animprovement over the MAD nasal nebuliser.

The results indicated that the Ari Mist and 180 kHz ultrasonicnebulisers gave comparable levels of delivery efficiency with between 20and 30% GFP positive cells detected. The cell viability was higher withthe Ari Mist head. Furthermore, this nebuliser is smaller, easier tohandle and does not require to be powered to operate. All these reasonscontributed to selection of a nebulizer, e.g., Ari Mist nebuliser, asspray head for soluporation and the optimal delivery parametersevaluated for mRNA delivery, e.g, as shown in Table 4.1. This set ofparameters was the result of a wide screening and became the startingpoint for a further study of refined optimisation whereby in addition tovolume, distance and length of spray other parameters such asconstituents of the delivery solution, cell number and filter plate werefinely tuned to further improve mRNA delivery. Table 4.1 shows a list ofparameters and the ranges tested including the preferred parameters fordelivery of mRNA (e.g., model cargo GFP mRNA) to T-cells. Table 4.1includes ranges tested and preferred parameters for both the benchtopFlexi (benchtop) and the Midi (scaled-up) systems.

Optimisation of the Steps for Application of Delivery Solution to CellsAtomiser Height

A number of parameters were assessed in order to increase uptakeefficiency and expression of mRNA in human primary T cells. The heightat which the AriMist atomiser was assessed to observe if an effect onGFP mRNA delivery to T cells existed. 1×10⁶ human primary T cells wereseeded per well in a 96-well filter plate (Pall; Supor, 1.2 μn; CAT#8039). The plate was centrifuged at 300×g for 5 min and the cellmonolayer was sprayed with 4 μl of delivery solution containing 0.57μg/μl of GFP mRNA. The atomiser height was assessed at 31, 26 and 11 mmabove the bottom of the well (a comparison of 26 mm vs 12 mm wasassessed in another experiment). Stop solution (50 μl) was applied aftera 2 minute incubation and 30 s later normal media (100 μl) was applied.The cells were incubated overnight at 37° C. and 5% CO₂ in a humidifiedincubator and assessed for GFP fluorescence by flow cytometry. Uptake ofmRNA by T cells was achieved when the atomiser was placed 31 and 26 mmabove the bottom of the well. In some cases, a percentage of the 4 μldelivered did not enter the well. A preferred height of 12 mm above thebottom of the well was chosen (FIG. 27A, B). This allowed accuratedosing of payload and prevented overspray contaminating other wells. Thereduction in height also allowed for a reduction in volume delivered.Range of height is between 11 mm and 31 mm above bottom of well(filter).

Volume Delivered

A comparison of volumes was undertaken to determine the optimal volumedelivered that would allow the greatest uptake of mRNA by T cells. 1×10⁶human primary T cells were seeded per well in a 96-well filter plate(Pall; Supor, 1.2 μm; CAT #8039). The plate was centrifuged at 300×g for5 min and the cell monolayer was sprayed with 4, 1 or 0.5 μl of deliverysolution containing 0.57 μg/μl of GFP mRNA. Stop solution (50 μl) wasapplied after a 2 minute incubation and 30 s later normal media (100 μl)was applied. The cells were incubated overnight at 37° C. and 5% CO₂ ina humidified incubator and assessed for GFP fluorescence by flowcytometry. An optimal volume of 1 μl per well was determined (FIG. 28).The Solupore test rig system allows the volume sprayed to be adjusted byaltering either the pressure applied to the ElveFlow or by the durationthat the valve remains open. For the first condition, the duration ofthe valve opening was set at 280 ms and the pressure was set at 70 mBar.The second condition reduced the valve opening time to 140 ms and thepressure set at 140 mBar. The optimal method was to reduce the valveopening time to 140 ms.

Tonicity of Delivery Solution

Previous experiments used a delivery solution that was hypotonic whencompared to the cell. An assessment of delivery solutions where thetonicity was altered by the further addition of KCl was conducted. Humanprimary T cells were seeded at 1×10⁶ per well in a 96-well filter plate(Pall; Supor, 1.2 μm; CAT #8039). The plate was centrifuged at 300×g for5 min and the cell monolayer was sprayed with 4 μl of delivery solutioncontaining 0.57 μg/μl of GFP mRNA. In the first condition, the deliverysolution contained 12.5 mM KCl resulting in a solution hypotonic to thecell. The second condition contained 106 mM KCl resulting in a solutionisotonic to the cell cytoplasm. Other concentrations between 10mM and500 mM, e.g., 12.5 mM KCl, 328 and 500 mM KCl, were also tested. Athigher tonicity, 328 and 500 mM KCl and GFP expression was demonstratedat a reduced level. Thus, the useful range is from 12.5 to 500 mM, e.g.,50-150 mM, e.g., 100-125 mM, e.g., 100-110 mM, with 106 mM being apreferred concentration of KCl.

Once the cells were sprayed, Stop solution (50 μl) was applied after a 2minute incubation and 30 s later normal media (100 μl) was applied. Thecells were incubated overnight at 37° C. and 5% CO₂ in a humidifiedincubator and assessed for GFP fluorescence by flow cytometry. Anoptimal concentration of 106 mM KCl, which was isotonic to the cell, wasdetermined (FIG. 29).

Multiple “hits”

Due to the gentle nature of the Solupore technology, the cells can beaddressed on numerous occasions, without a drop in the cell viability orfunctionality. An assessment of the preferred number of “hits”(treatments) was undertaken with, e.g., a 1-, 2- and 3-hit strategy.Human primary T cells were seeded at 1×10⁶ cells per well in a 96-wellfilter plate (Pall; PES, 1.2 μm). The plate was centrifuged at 300×g for5 min and the cell monolayer was sprayed with 1 μl of delivery solutioncontaining 0.57 μg/μl of GFP mRNA. Once the cells were sprayed, Stopsolution (50 μl) was applied after a 2 minute incubation and 30 s laternormal media (100 μl) was applied. For the 2-hit strategy the cells wereincubated for 2 hours before the spray process was repeated and the3-hit strategy had a further repeat after a 2 hour incubation. Beforeeach additional hit, and at the end of the 2 hr incubation, the wellscontaining the cell suspension were sealed using a film (e.g., ParafilmM) and the plate was placed on top of an agitator, e.g., a vortex mixer,and held for 15 s. The cell suspension was then pipette mixed 3 times.The vibration from the vortex and the mixing enabled the orientation ofthe cells to be “shuffled” prior to the subsequent hits. Cells wereincubated overnight at 37° C. and 5% CO₂ in a humidified incubator andassessed for GFP fluorescence by flow cytometry. The 3-hit strategyappeared to be optimal when looking at uptake, viability and cell yield(FIG. 30).

Cell Seeding Density

An assessment of the optimal T cell seeding density using the AgilentPCTE plate was undertaken. Human primary T cells were seeded at 1.25,2.5, 3.5, 5 and 7.5×10⁵ cells per well in a 96-well filter plate(Agilent; PCTE, 0.4 μm). The plate was centrifuged at 350×g for 2 minand the cell monolayer was sprayed with 1 μl of delivery solutioncontaining 0.57 μg/μl of mRNA. Once the cells were sprayed, Stopsolution (50 μl) was applied after a 2 minute incubation and 30 s laternormal media (100 μl) was applied. The cells were incubated for 2 hoursbefore the process was repeated. At the end of this spray the cells wereincubated overnight at 37° C. and 5% CO₂ in a humidified incubator andassessed for GFP fluorescence by flow cytometry. The seeding density of3.5×10⁵ cells was shown to be optimal (FIG. 31). The average T cell sizefollowing Dynabead activation is ˜9.5 μm (70.9 μm²; FIG. 32). To confirmthe seeding density, the density based on the average diameter of anactivated T cell and the area of the addressable area of the filter well(19.6 mm²) was calculated. From the calculation the number of cells thatwould form a monolayer on the filter is approximately 2.77×10⁵.

Masking

It was noted previously that a region of negative cells existed aroundthe edge of the well. It is possible that this edge-effect exists due toeither (or combinations of): poor spray targeting; increased volumearound edge due to meniscus; or ineffective droplet impact at edge;pressure turbulence at the edges. A strategy to overcome the edge-effectwould be to produce a seeding mask that would be present during seedingand removed after centrifugation which would prevent cells from beingseeded close to the well edge. To test this theory the masks were placedinto the PCTE plate wells reducing the diameter of the well from 5.2 to4 mm 2.5×10⁵ cells human T cells were seeded within the mask and 3.5×10⁵unmasked wells as a control. The plate was centrifuged at 350×g for 2min and the masks were removed before spraying the cells. This meantthat cells were seeded only up to about 0.5 mm from the walls of thewell. The cells were sprayed with delivery solution containing 0.57μg/μl of GFP mRNA using a 1-hit strategy. At the end of the process thecells were incubated overnight at 37° C. and 5% CO₂ in a humidifiedincubator and assessed for GFP fluorescence by flow cytometry. Theresults showed that the sample from the masked wells gave 67.7% uptakebut the samples without the mask had 53.1% uptake, suggesting that thereis an edge effect and that by having the mask present for seedingnegates this (FIGS. 60A, B). Thus, optionally cells are prevented frombeing seeded up to the edge of the well, thereby leading to an increasein transfection efficiency.

Force

To examine the correlation between force exerted from the spray anduptake, force sensor analysis was conducted at different heights andpressures to establish a baseline of results. The pressure that drivesthe air through the atomizer was adjusted from 0.5 to 2 Bar and theforce experienced at the bottom of the well was measured using a forcesensor. The height was also adjusted to 31, 26 and 11 mm. The resultsindicated that air pressure was the single largest factor that affectsthe force exhibited by the spray with a small drop in force at the lowerheight of 11 mm With a force of 2.0 bar at 11 mm, the same force isexperienced with the larger heights and the normal pressure of 1.65 bar(FIG. 55). This experiment was repeated with a more robust analysis ofwhat affects the force exerted by the spray. Again, the air pressure wasthe greatest contributing factor to force exerted by the spray (FIG.56). At lower pressures, the force experience at different heightsbecomes negligible with force at 1.15 bar at heights of 11, 26 and 31 mmbeing indistinguishable. At 2.15 bar, the force at the 26 and 31 mmheight was greater than the 11 mm This can be explained with the factthe larger height with higher pressure drags more air from theatmosphere into the spray path thus increasing the mass of the spray,due to force=mass×acceleration. The same parameters from FIG. 4.11 werethen tested to correlate the force profile with GFP mRNA uptake in CD3+T-Cells. 1×10⁶ human primary T cells were seeded per well in a 96-wellfilter plate (Pall; Supor, 1.2 μm; CAT #8039). The plate was centrifugedat 300×g for 5 min and the cell monolayer was sprayed with 4, 2, 1 or0.67 μl of delivery solution containing 0.57 μg/μl of GFP mRNA. Stopsolution (50 μl) was applied after a 2 minute incubation and 30 s laternormal media (100 μl) was applied. The cells were incubated overnight at37° C. and 5% CO₂ in a humidified incubator and assessed for GFPfluorescence by flow cytometry. The GFP expression results indicatedthat GFP mRNA uptake can be achieved under a wide range on conditionsvarying in delivery volume, distance and air pressure (FIG. 57). Forceexperienced by the spray does not directly correlate with uptake. Whilethere may be a minimum force required to achieve uptake and a maximumbefore we see a loss in viability, this range is broad (1-2 bar).

Ranges of conditions suitable for delivery of mRNA to non-adherentcells, e.g., T cells such as primary human T cells, are summarizedbelow.

Benchtop Flexi - Range Midi - Range Spray Atomiser Any Nebuliser AnyNebuliser Distance 2-60 mm 10-300 mm Pressure 0.1-6.0 Bar 0.1-6.0 BarSpray duration 50-3000 ms/ 50-3000 ms/ (air/liquid) 50-2900 ms 50-2900ms Flow rate 50-1000 μl/min 2000-100,000 μl/min Delivery solution KClConcentration 5-1000 mM 5-1000 mM Ethanol 5-50% 5-50% Volume sprayed 0.2to 100 μl 20-3000 μl mRNA/hit 0.1-75 ug 0.1-2250 ug Hits Number of hits1-10 1-10 Shuffle between hits No/Yes Yes Monolayer formationpossibilities Seeding density 0.25-10 × 10⁴ per mm² 0.25-10 × 10⁴ permm² Vacuum 5 s-300 s @ −10-−1000 n.d. mBar Centrifugation 30 s-5 min @n.d. 100-1000 x g Positive pressure 5 s-300 s @ 10-1000 5 s-300 s @10-1000 mBar mBar Filter type/pore size PES/PCTE PES/PCTE/PETE 0.1-8.0μm 0.1-8.0 μm

The preferred, e.g., optimal, conditions for delivering mRNA to T cellsis outlined in Table 4.1

TABLE 4.1 Conditions for mRNA delivery to T cells over time. BenchFlexi - Bench Flexi - Midi - Midi - Optimal Ranges Optimal Ranges SprayAtomiser AriMist MAD Nasal/ LB-100 LB-100/T-2100 Ultrasonic emitters(60-180 kHz)/ Burgener Research Nebulizers (AriMist/PEEKMiraMist/PFA250/X- 175/1-2100) Distance 12 mm 11-31 mm 82 mm 62-140 mmPressure 1.65 Bar 0.5-3.0 Bar 2.5 Bar 1-6 Bar Spray duration 240 ms/140ms 130-1000 ms/50-900 ms 520 ms/420 ms 380-800 ms/280-700 ms(air/liquid) Flow rate 7.14 μl/s 21.2-7.14 μl/s 238 μl/s 100-500 μl/sDelivery Solution KCl Concentration 106 mM 12.5-106 mM 106 mM 12.5-106mM Ethanol 27% 20-30% 24% 20-30% Volume sprayed 1 μl 0.5 to 10 μl 100 μl30-300 μl mRNA/hit 0.57 μg 0.57-2.28 μg 10.0 μg 3-60 μg Hits Number ofhits 2 1-4 2 1-2 Shuffle between hits Yes No/Yes Yes Yes Monolayerformation possibilities Seeding density 1.8 × 10⁴ per mm² 1.3-4 × 10⁴per mm² 1.5 × 10⁴ per mm² 1.3-4 × 10⁴ per mm² Vacuum n.d. 10 s-120 s @−10-150 mBar n.d. n.d. Centrifugation 2 min @ 350 g 30 s-5 min @ 150-350x g n.d. n.d. Positive pressure 45 s @ 100 10s-120 s 10-150 40 s @ 12020-80 s @ 100-200 mbar mBar mbar mBar Filter type/pore size PCTE/0.4 μmPES/PCTE PCTE/0.4 μm PES/PCTE/PETE 0.4-3.0 μm 0.2-5.0 μm

Transfection Comparison

Delivery and viability compared with electroporation—electroporation isa widely used method for vector-free intracellular delivery. Therefore,delivery efficiency and cell viability levels using the delivery methodof the current subject matter was compared with electroporation.

When 3 μM of model payload, 10 kDa dextran-Alexa488, was delivered toA549 cells using the current subject matter technology, deliveryefficiency was 52.8% (+/−2.7%) compared with 92.9% (+/−0.6%) forelectroporation (FIGS. 33A, B, C). The percentage of cells that survivedthe delivery process was analysed by propidium iodide exclusion and flowcytometric analysis. For the current subject matter technology, cellsurvival compared with untreated control cells was 78.3% (+/−4.1%)compared with 73.0% (+/−9.8%) for electroporation (FIGS. 33A, B, C).

For most delivery methods, effective delivery must be balanced withmaintenance of cell viability. In order to examine this balance, atransfection score ((transfected cells/total cells)x(viable cells/totalcells)) was used to obtain an aggregate characterisation of cell loss,cell viability and transfection efficiency for the current subjectmatter delivery technology compared with electroporation. A score of 1.0would indicate 100% transfection efficiency, 100% cell viability andthat no cells were lost during the procedure. The transfection score forthe current subject matter technology was 0.33 (+/−0.05) and forelectroporation was 0.51 (+/−-0.13) with no significant differencebetween the scores (FIG. 33C).

A comparison of Solupore and Nucleofection (4D; Lonza) delivery of mRNAto human T cells was undertaken to benchmark the current technology. Theamount of mRNA delivered (μg) was matched per cell. For Soluporation,human primary T cells were seeded at 1×10⁶ cells per well in a 96-wellfilter plate (Pall; PES, 1.2 μm). The plate was centrifuged at 300×g for5 min and the cell monolayer was sprayed with 1 μl of delivery solutioncontaining 0.57 μg/μl of GFP mRNA. Once the cells were sprayed, Stopsolution (50 μl) was applied after a 2 minute incubation and 30 s laternormal media (100 μl) was applied. The cells were incubated overnight at37° C. and 5% CO₂ in a humidified incubator and assessed for GFPfluorescence by flow cytometry. For Nucleofection, 5×10⁶ human primary Tcells were washed in PBS and resuspended in 40 μl P3 buffer containing 2μg GFP mRNA (Lonza). The cells were then added to the nucleocuvettestrip and nucleofected as per instructions. 100 μl media was added toeach well and transferred to recovery flask containing 10 mls media. Thecells were incubated overnight at 37° C. and 5% CO₂ in a humidifiedincubator and assessed for GFP fluorescence by flow cytometry. GFP mRNAexpression levels were 40.3% and 89.3%, respectively (FIG. 34). Theaverage median fluorescent intensity from 5 experiments and 2 donors was203,059 and 113,895, respectively (FIGS. 35A, B, C, D). A dose responseof mRNA delivered by each technology is shown in FIG. 36.

Endocytosis Independent

Diffusion of cargo into cells and resealing of plasma membrane. Havingdemonstrated the ability of this method to deliver a broad range ofcargoes to a range of cells types, the mechanism of cargo uptake intocells and the reversal of the cell permeability was examined Alimitation of other delivery techniques is their dependence on activeuptake pathways such as endocytosis which can lead to sequestration ofthe cargo rendering it unavailable to function in the cell. For example,liposome-mediated delivery involves both clathrin- and caveolar-mediatedendocytosis (Cui S, Wang B, Zhao Y, Chen H, Ding H, Zhi D, et al.Transmembrane routes of cationic liposome-mediated gene delivery usinghuman throat epidermis cancer cells. Biotechnol Lett. 2014;36(1):1-7.doi: 10.1007/s10529-013-1325-0. PubMed PMID: 24068499; PubMed CentralPMCID: PMCPMC3889874.) while iTOP delivery involves macropinocytosis(D'Astolfo D S, Pagliero R J, Pras A, Karthaus W R, Clevers H, Prasad V,et al. Efficient intracellular delivery of native proteins. Cell. 2015;161(3):674-90. doi: 10.1016/j.ce11.2015.03.028. PubMed PMID: 25910214.).

During the experiments using Soluporation, immediate uptake of cargointo cells was observed. Using 10 kDa dextran-FITC as a model cargo,within 30 sec of applying the delivery solution, before Stop solutionwas added, cargo was visible within the cells (FIGS. 37A, B, C, C). Therapid influx of cargo into the cells indicates that it is unlikely thatdelivery involves endocytosis. The results showing loading of a widerange of molecular species into a range of cell types indicates that adiffusion mechanism through the cell membrane is the mechanism of entryof macromolecules into the cells. To test the contribution of alternateuptake mechanisms such as active pathways and internalization inendocytotic vesicles, A549 cells were pretreated with Dynasore (4 mM) orchloropromazine (20 μM) to inhibit clathrin-mediated endocytosis orNystatin (20 μg/ml) or EIPA (100 μM) to inhibit caveolar-mediatedendocytosis and micropinocytosis respectively. Expression of EGFP mRNAremained unchanged in the presence of these inhibitors indicating thatthis method results in direct delivery into the cytoplasm of cells anddoes not rely on endocytosis (FIG. 37C). Furthermore, in addition tofollowing the procedure reported by D'Astolfo et al. (D'Astolfo et al.2015), Lipofectamine 2000 was included as a positive control to confirmDynasore-mediated inhibition of clathrin-mediated endocytosis (FIG.37C).

It was noted that the delivery method was very gentle on cells withlittle if any cell death or damage evident. The method allows thepermeabilised plasma membrane to reseal rapidly, hence retaining highlevels of cell viability. To examine the rate of recovery of the cellmembrane after permeabilization, delivery solution was applied to A549cells in the absence of cargo. At subsequent time points (0 to 182.5min), this delivery solution was removed and 50 μl PBS containingpropidium iodide (PI) (100 μg/ml) is added. After 2 min incubation, thePI solution was removed and the cells were harvested. PI uptake wasanalysed by flow cytometry. For basal levels of PI uptake, untreatedcells received 50 μl PI in PBS. The results demonstrate that the cellsremain permeable to PI for several minutes but reseal over a period of 6min post treatment (FIG. 37C). After 6 minutes there is no furtheruptake. Thus not only do the cells load within 2 minutes of exposure tothe delivery solution, but the membrane has effectively recovered itsintegrity within 6 minutes of beginning the procedure. These dataindicate that delivery of agents using Soluporation does not involveendocytosis.

Gene Editing in T Cells

In order to demonstrate another functional output of T cells followingdelivery of cargo, gene editing of T cells was assessed usingCRISPR/Cas9 RNP delivery.

CRISPR/Cas9 RNP Delivery. A two guide RNA strategy was employed toknockdown the PDCD1 gene

[ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA] (SEQ ID NO:1), which encodes for the PD-1protein.

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTS SPARRGSADGPRSAQPLRPEDGHCSWPL (PD1-1protein amino acid sequence, SEQ ID NO:2). Equimolar amounts ofcrisprRNA (a mixture of both GCGTGACTTCCACATGAGCG (SEQ ID NO:3) andGCAGTTGTGTGACACGGAAG (SEQ ID NO:4) crisprRNAs, also equimolar amounts;(Su, S. et al. CRISPR-Cas9 mediated efficient PD-1 disruption on humanprimary T cells from cancer patients. Sci. Rep. 6, 20070; doi:10.1038/srep20070 (2016)) and tracrRNA were incubated for 10min at RT. 5μg Cas9 (IDT) was then added so that the final molar ratio of Cas9 toguide RNA was 1:3 and incubated for a further 10 min at RT. Cas9 RNPs(in buffer, with α-crystallin (220 μM) and Ethanol (25% v/v)) weredelivered to T cells by using the vector-free intracellular deliverymethod described. Using the vector-free delivery method describedherein, RNPs were delivered to 1.5×10⁶ cells per treatment and PD-1expression was analysed at 72 h post-transfection.

Cell viability. FACS Sample Preparation and Analysis: Cell viabilityfollowing the vector-free intracellular delivery method, delivery wasassessed using 7-AAD viability staining solution (Sigma). Cells were inwashed in PBS+1% fetal bovine serum (FACS buffer) followed by incubationwith 7-AAD (1:40 for 5-10 min protected from light at room temperature),followed by resuspension in PBS+1% FBS (FACS buffer). PD-1 labelling wascarried out using APC-conjugated anti-human CD279 (PD-1) (Biolegend) andprocessed on the BD Accuri C6 flow cytometer (Becton Dickinson, USA).Data was analysed using the C6 software. Cell debris was excluded fromwhole cells using forward and side scatter parameters. Single cells wereselected by excluding doublets in the FSC height vs FSC are plot. GFPexpression was analyzed on gated viable cells.

Knock-down of immune check point gene expression such as PD-1 expressionin T cells following vector-free delivery of CRISPR/Cas9 RNPs

CRISPR/Cas9 RNPs targeting the PDCD1 gene were delivered to T cellsusing either the vector-free intracellular delivery method describedherein or by electroporation. PD-1 expression was analysed by flowcytometry 72 h post-transfection.

Delivery of CRISPR/Cas9 gene editing tools to activated T cells resultedin a 28% reduction in PD-1 expression by the vector-free intracellulardelivery method described herein compared with 53% inhibition inelectroporated cells (FIGS. 38A, B).

The proliferative capacity of gene-edited cells was assessed over a4-day period by observing the formation of T cell aggregates in culture.Following delivery of RNPs, cells were returned to culture. After 4days, cells were observed using a microscope. Cell aggregation in thevector-free intracellular method-transfected cells was similar tountreated control activated cells indicating that they were not affectedby the vector-free intracellular delivery method described. In contrast,the growth rate of electroporated cells was significantly affected asindicated by significantly fewer cell aggregates (FIG. 39)

CAR-T: Expression of Chimeric Antigen Receptor (CAR) in Primary T CellsFollowing mRNA Delivery

mRNA generated from a commercially sourced CD19 CAR plasmid wassuccessfully delivered to human-derived activated T cells. Surfaceexpression of the CAR was detected by flow cytometry with up to 50% ofthe population positive for CAR expression.

Materials and Methods

Cell culture. Human peripheral blood mononuclear cells (PBMC) wererecovered by centrifugation over a Percoll gradient from Leuko Pak(AllCells Alameda, CA). CD3⁺ enriched lymphocytes were isolated bymagnetically activated cell sorting using CD3 Microbeads (Miltenyi).Cells were cryopreserved in 10% dimethyl sulphoxide (DMSO) and foetalbovine serum (FBS). Following initial thawing from stock aliquots, CD3⁺T cells were cultured in human recombinant interleukin-2 (IL-2) withprimary and co-stimulatory antibody activation using various protocols(see below) in a humidified tissue culture incubator at 37° C. and 5%CO_(2.)

Construction of CD19 CAR Plasmid

A CD19 CAR plasmid was sourced commercially (Creative Biolabs, NY, USA)and mRNA generated from the plasmid. The full length of chimeric antigenreceptor (CAR) was synthesized and subcloned into lentivirus vector. Theinsert was confirmed by Sanger sequencing. The structure of CAR vectoris illustrated in FIG. 86. The amino acid sequence of scFv (Anti-CD19scFv VL-Linker-VH) is depicted in FIG. 87A. The nucleotide sequence(codon optimized) of the CAR cassette is depicted in FIG. 87B. The aminoacid sequence of the CAR cassette is depicted in FIG. 87C. TheRestriction Digestion map is depicted in FIG. 88. The Quality Controlresults of the vector design is depicted in FIG. 89. The CAR sequencealignment validation is depicted in FIG. 90. The sequence alignmentresults showed that the sequence of the constructed plasmid was inaccordance with the design.

CAR mRNA Delivery

4 μg of mRNA (from CD19 CAR plasmid described above) was added to Bufferand Ethanol (27% v/v) also added and delivered to 1.5×10⁶ T cells usingtechnology described herein to 1.5×10⁶. 24 hr later the cells wereharvested and analysed for mRNA expression using flow cytometry.

Flow Cytometry

Biotinylated protein L (AcroBiosystems) was reconstituted in phosphatebuffered saline (PBS) at 1 mg/ml. For FACS staining, 1×10⁶ cells wereharvested and washed three times with ice cold PBS containing 4% bovineserum albumin (BSA) wash buffer. After wash, cells were resuspended in0.2 ml of the wash buffer and incubated with 1 μg of Protein L for 45minutes at 4° C. Cells were washed a further three times and thenincubated in the dark with 10 μl of PE-conjugated streptavidin in 0.2 mlof wash buffer. To assess cell viability following the vector-freedelivery method described herein, 7-AAD (Sigma) was used to stain thecells. Briefly, cells were in washed in PBS+1% fetal bovine serum (FACSbuffer) followed by incubation with 7-AAD (1:40 for 5-10 min protectedfrom light at room temperature), followed by resuspension in PBS+1% FBS(FACS buffer). Samples were processed on the BD Accuri C6 flow cytometer(Becton Dickinson, USA) and data was analysed using the C6 software.Cell debris was excluded from whole cells using forward and side scatterparameters. Single cells were selected by excluding doublets in the FSCheight vs FSC are plot. CAR-T expression was analysed on gated viablecells.

Untransfected cells were used as untreated controls (UT). A shift influorescence intensity was observed using the delivery method fortreated cells was indicative of CAR expression following mRNA delivery(FIG. 45).

Assessment of Functionality of T Cells Post-Soluporation

In order to demonstrate that the functionality of T cells transfectedusing Solupore technology is equivalent to or better than cellstransfected using electroporation, e.g., with the Neon electroporator,and nucleofection, e.g., using the Lonza 4D nucleofector. Cell membraneprotein expression, gene expression, cell proliferation rate, in vitrofunctionality and in vivo functionality assays were carried out.

(i) T Cell Membrane Protein Expression Analysis

The goal of this study was to determine whether expression of cellsurface proteins on T cells was affected by the soluporation process andto compare with electroporation and nucleofection.

Method: Activated T cells were seeded at 1.5×10⁶ cells per well of a96-well filter plate (Acroprep, 1.2 μm Supor membrane; Pall, USA). Mediawas removed from the wells by centrifugation at 300×g for 5 min. 7 μl ofdelivery solution (32 mM sucrose, 12 mM potassium chloride, 12 mMammonium acetate, 5 mM HEPES and 27% ethanol in molecular grade water(all from Sigma-Aldrich)) containing 4 μg GFP mRNA was then sprayed intoeach well using the vector-free delivery spray instrument. Followingdelivery, the cells were incubated in this solution for 2 minprior tothe addition of 50 μl Stop Solution (0.5× PBS). Thirty seconds later Tcell media was added (100 μl) and cells were allowed to recover at 37°C. and 5% CO₂ overnight. For electroporation and nucleofection, 5×10⁶cells and 2.5×10⁶ cells were used respectively per transfection. GFPexpression and viability were assessed at 6 h and 24 h post-delivery.

The expression of cell surface CD4 and CD8 was examined at 6 hr and 24hr after either nucleofection, electroporation or Soluporation. At 6 hrpost-transfection, the percentage of T cells expressing CD4 and CD8 wasunchanged compared to untreated control cells for each transfectionmethod. However, at 24 hr, expression was significantly reduced inelectroporated cells whereas expression in soluporated cells andnucleofected cells was similar to control untreated cells (FIG. 47A, B).

(ii) Global mRNA Expression Analysis

The goal of this study was to obtain a molecular signature of cellularperturbation induced by the soluporation process, and to compare thatsignature with electroporation and nucleofection. 16 samples wereanalysed using mRNA microarrays: two donor T cells were taken, samplesincluded an untreated control, neon, nucleofector and Soluporetransfected cells with GFP-mRNA, at 6 hr and 24 hr time points.

Method: Activated T cells were seeded at 1.5×10⁶ cells per well of a96-well filter plate (1.2 μm PES membrane; Pall, USA). Media was removedfrom the wells by centrifugation at 300×g for 5 min. 1 μl of deliverysolution (32 mM sucrose, 12 mM potassium chloride, 12 mM ammoniumacetate, 5 mM HEPES and 27% ethanol in molecular grade water (all fromSigma-Aldrich)) containing 0.2 μg GFP mRNA was then sprayed into eachwell using the vector-free delivery spray instrument. Following thespray, cells were incubated in this solution for 2 min prior to theaddition of 50 μl Stop Solution (0.5× PBS). Thirty seconds later T cellmedia was added (100 μl). A second spray was carried out 2 hrs after thefirst. Cells transferred to an incubator at 37° C. and 5% CO₂. Forelectroporation and nucleofection, 5×10⁶ cells and 2.5×10⁶ cells wereused respectively per transfection. GFP expression and viability wereassessed at 6 h and 24 h post-delivery.

The highest level of gene expression changes occurred in Neonelectroporation treatments. However, a drawback of electroporation isreduced viability and functionality of the treated cellspost-electroporation. Of the 20,893 mRNAs analysed, Neon electroporationhad a combined total of 317 changed over all timepoints (6 hr and 24 hr)and both donors, Solupore had 32 changed (Tables 5.1 and 5.2) andnucleofector had 24 changed (Tables 5.3 and 5.4; FIG. 48A, B, C).Notably, the background level of false positives for this microarrayanalysisis approximately 10%. For Solupore and nucleofection, the numberof genes changed was only slightly above this threshold, demonstratingthat Solupore and Nucleofector cause a minimal level of perturbation tocell gene expression. The high level of gene expression changesindicates that the Neon electroporation process perturbs the cells morethan Soluporation of nucleofection. Cell perturbation is undesirable;therefore, the data indicate that Neon electroporation is less desirableas a transfection method compared with Soluporation and nucleofection.Soluporation is associated with several significant advantages comparedto electroporation and nucleofection. Such advantages include high levelreliable delivery of cargo, e.g., mRNA, to primary human cells, whilepreserving the integrity, function, and proliferation capabilities ofthe cells treated.

TABLE 5.1 List of gene expression changes in soluporated cells comparedwith untreated control cells at 6 hr post transfection. (Numbersindicate fold change compared with untreated control cells.) FOSB FosBproto-oncogene, AP-1 transcription factor subunit OsteoclastdifferentiationIL-1 2.673 DMTF1 cyclin D binding myb like transcriptionfactor 1 — 1.954 ATF3 activating transcription factor 3 HTLV-I infection2.983 SLITRK5 SLIT and NTRK like family member 5 — −2.155 PPIL6peptidylprolyl isomerase like 6 — 2.34 ARRDC4 arrestin domain containing4 — 2.602 TSC22D3 TSC22 domain family member 3 — 2.858 FSD1L fibronectintype III and SPRY domain containing 1 like — 2.377 SNX9 sorting nexin 9— 1.863 PPP1R15A protein phosphatase 1 regulatory subunit 15A Proteinprocessing in endoplasm 2.033 — — — 1.715 PIFO primary cilia formation —1.816 RASGEF1B RasGEF domain family member 1B — 2.622 TMEM154transmembrane protein 154 — 1.869 TTC30B tetratricopeptide repeat domain30B — 2.734 TCP11L2 t-complex 11 like 2 — 3.804 KLF6 Kruppel like factor6 — 1.674 — — — −1.644 CCDC173 coiled-coil domain containing 173 — 1.974— — — 2.008 IL1RN interleukin 1 receptor antagonist — 2.021 NPIPB3nuclear pore complex interacting protein family member B3 — 1.77 GPCPD1glycerophosphocholine phosphodiesterase 1 Glycerophospholipid metabolic1.967 MGEA5 meningioma expressed antigen 5 (hyaluronidase) Insulinresistance 1.811 ZC2HC1A zinc finger C2HC-type containing 1A — 1.762EPC2 enhancer of polycomb homolog 2 — 1.803 NPIPB11 nuclear pore complexinteracting protein family member B1 — 1.772 ZNF440 zinc finger protein440 — 1.653The data indicate that only a low number (e.g., negligible) of changesin gene expression occur in soluporated cells at 6 hr post transfection.

TABLE 5.2 List of gene expression changes in soluporated cells comparedwith untreated control cells at 24 hr post transfection. Numbersindicate fold change compared with untreated control cells. SymbolDescription KEGG Pathways FC CXCL13 C-X-C motif chemokine ligand 13Cytokine-cytokine receptor 2.525 FAM198B family with sequence similarity198 member B — 2.337 — — — −1.918 GPM6A glycoprotein M6A — 1.925 — — —2.013 GTF2H5 general transcription factor IIH subunit 5 Basaltranscription factors 1.796 — — — 1.881 TREML2 triggering receptorexpressed on myeloid cells life — 1.882 HFE2 hemochromatosis type 2(juvenile) — −1.998 SCUBE3 signal peptide, CUB domain and EGF likedomain — −1.77 TCTEX1D2 Tctex1 domain containing 2 — 1.825 WLS wntlessWnt ligand secretion mediator — 1.713 — — — −1.766 — — — −1.859 — — —1.797 CNTN5 contactin 5 — −1.706 ZFP2 ZFP2 zinc finger protein — −1.883SNX9 sorting nexin 9 — 1.738 — — — 1.891 — — — 1.75 TSSK4 testisspecific serine kinase 4 — 1.829 ADAMTS6 ADAM metallopeptidase withthrombospondin t — −1.725 WIPI1 WD repeat domain, phosphoinositideinteracting Autophagy - otherAutopha 1.655 — — — −2.062 OSBPL10oxysterol binding protein like 10 — −1.817 — — — −1.782 UBASH3Aubiquitin associated and SH3 domain containing — 1.703 — — — 1.757 — — —1.654 OR52A5 olfactory receptor family 52 subfamily A member Olfactorytransduction −1.632 SMCO4 single-pass membrane protein with coiled-coild — −1.647 C19orf38 chromosome 19 open reading frame 38 — −1.776 WDR19WD repeat domain 19 — 1.834The data indicate that only a low number (e.g., negligible) of changesin gene expression occur in soluporated cells at 24 hr posttransfection.

TABLE 5.3 List of gene expression changes in nucleofected cells comparedwith untreated control cells at 6 hr post transfection. Numbers indicatefold change compared with untreated control cells. EFHC2 EF-hand domaincontaining 2 — 2.883 NEK11 NIMA related kinase 11 — 2.206 QPCTglutaminyl-peptide cyclotransferase — −2.059 — — — 1.78 RNF125 ringfinger protein 125 RlG-I-like receptor signaling . . . −2.219 — — —1.763 GJB2 gap junction protein beta 2 — 1.827 PIFO primary ciliaformation — 1.801 FSD1L fibronectin type III and SPRY domain containing1 like — 2.099 LOC654841 uncharacterized LOC654841 — 1.92 ADGRG1adhesion G protein-coupled receptor G1 — 1.836 — — — 1.631 CDKL2 cyclindependent kinase like 2 — 1.701 LY96 lymphocyte antigen 96 NF-kappa Bsignaling pathwayTol 1.975 APBB1IP amyloid beta precursor proteinbinding family B member Rap1 signaling pathwayPlatelet a 1.643

The data indicate that only a low number (e.g., negligible) of changesin gene expression occur in nucleofected cells at 6 hr posttransfection.

TABLE 5.4 List of gene expression changes in nucleofected cells comparedwith untreated control cells at 24 hr post transfection. Numbersindicate fold change compared with untreated control cells. GJB2 gapjunction protein beta 2 — 2.446 CMPK2 cytidine/uridine monophosphatekinase 2 Pyrimidine metabolism 2.056 WLS wntless Wnt ligand secretionmediator — 1.994 BEX5 brain expressed X-linked 5 — −2.038 — — — 1.927IL17F interleukin 17F Cytokine-cytokine receptor int . . . IL-17signaling pathwayTh −1.818 OSBPL10 oxysterol binding protein like 10 —−2.018 — — — −1.788 SYNGR3 synaptogyrin 3 — −1.793 TET2 tetmethylcytosine dioxygenase 2 — −1.9 — — — 1.875 NLRP7 NLR family pyrindomain containing 7 NOD-like receptor signaling pa . . . −1.963 NAPB NSFattachment protein beta — 2.417 IRF1 interferon regulatory factor 1Prolactin signaling pathwayPertussisHepatitis CHuman pap 2.265 IL2interleukin 2 Cytokine-cytokine receptor int . . . PI3K-Akt signalingpathway −2.421 ARMC4 armadillo repeat containing 4 — 1.714 MRC1 mannosereceptor, C type 1 PhagosomeTuberculosis 1.88 — — — −1.717 RPS6KC1ribosomal protein S6 kinase C1 — 2.005 — — — −1.64 BEND3 BEN domaincontaining 3 — 1.751 IL13RA1 interleukin 13 receptor subunit alpha 1Cytokine-cytokine receptor int . . . Jak-STAT signaling pathway 1.715DEFB118 defensin beta 118 — −1.788 SERGEF secretion regulating guaninenucleotide exchange family — −1.671 USP37 ubiquitin specific peptidase37 — −1.634 SOS1 SOS Ras/Rac guanine nucleotide exchange factor 1 MAPKsignaling pathwayErbB signaling pathwayRas signaling 1.752 — — — 1.948LOC729970 hCG2028352-like — 1.63

The data indicate that only a low number (e.g., negligible) of changesin gene expression occur in nucleofected cells at 24 hr posttransfection.

(iii) Cell Proliferation Analysis

For therapeutic applications, it is necessary that T cells are able toproliferate following modifications. Therefore, the ability of T cellsto proliferate following soluporation, electroporation and nucleofectionwas examined. The cells capacity to proliferate post-cryopreservationand thaw was also tested.

Method: Activated T cells were seeded at 1.5×10⁶ cells per well of a96-well filter plate (Acroprep, 1.2 μm Supor membrane; Pall, USA). 1 μlof delivery solution containing 0.2 μg GFP mRNA was then sprayed intoeach well. A second spray was carried out 2 hrs later. Forelectroporation and nucleofection, 5×10⁶ cells and 2.5×10⁶ cells wereused per transfection. Cells were transferred to an incubator at 37° C.and 5% CO₂. The next day, cells were harvested and counted. Cells werethen re-seeded at 0.5×10⁶/ml by adding additional media+IL-2 each dayfor 7 days. In another experiment, cells were cryopreserved in 10% DMSOand foetal bovine serum 24 hrs post-transfection. Cells were thawed andseeded at 0.5×10⁶/ml on day 0 in Immunocult media+IL-2. Cells werecounted and re-seeded by adding additional media each day for 5 days.

T cells were transfected and cells were counted each subsequent day for7 days. Proliferation rates in soluporated and nucleofected cells weresimilar to untreated control cells whereas the ability of Neonelectroporated cells was reduced compared with control cells (FIG. 49).Proliferation was unaffected by Soluporation post-cryopreservation andsubsequent thaw (FIG. 50). These data indicate that a significantdrawback of electroporation and/or nucleofection is a cell proliferationstall. A significant advantage of the Solupore system is the absence ofa cell proliferation stall.

(iv) Interferon-Gamma (IFNg) Secretion Analysis

For therapeutic applications, it is necessary that T cells are able toproduce IFNg following modifications. Therefore, the ability of T cellsto produce IFNg following soluporation, electroporation andnucleofection was examined Two different activation methods wereexamined, phorbol myristate acetate/ionomycin (PMA/I) and Dynabeads. Thecells ability to secrete IFN-γ was also tested post-cryopreservation andthaw following soluporation.

Method: Activated T cells were seeded at 1.5×10⁶ cells per well of a96-well filter plate (Acroprep, 1.2 μm Supor membrane; Pall, USA). 1 μlof delivery solution containing 0.2 μg GFP mRNA was then sprayed intoeach well. A second spray was carried out 2 hrs later. Forelectroporation and nucleofection, 5×10⁶ cells and 2.5×10⁶ cells wereused respectively per transfection. Cells were allowed to recover at 37°C. and 5% CO₂. The next day, cells were harvested and counted. Cellswere then re-seeded at 0.5×10⁶ /ml by adding additional media+IL-2 eachday for 7 days, after which the cells were allowed to return to aresting state by monitoring cell size. This was approximately 2 weeksafter initial activation. Cells were then re-stimulated with eitherDynabeads or a PMA/Ionomycin cocktail for 4 hrs, after which thesupernatants were recovered and stored at −20° C. until cytokineanalysis. IFN-γ ELISA (Biotechne) were carried out on all samples. Inanother experiment, Soluporated, nucleofected and electroporated cellswere harvested and counted 24 h after transfection. Cells were thenre-seeded at 0.5×10⁶/ml by adding additional media+IL-2 each day for 7days, after which the cells were allowed to return to a resting state bymonitoring cell size. This was approximately 2 weeks after initialactivation. Cells were then re-stimulated with either Dynabeads or aPMA/Ionomycin cocktail for 4 hrs, after which the supernatants wererecovered and stored at −20° C. until cytokine analysis. IFN-y ELISA(Biotechne) were carried out on all samples.

IFNg production was not reduced in T cells following soluporation,nucleofection or electroporation compared with control cells (FIG. 51).Freshly thawed soluporated cells did not lose their capacity to secreteIFN-γ when compared to untreated controls (FIG. 52).

(v) In Vivo Engraftment in T Cells Following Delivery of 3 kDa Dextran

In order to determine the effect of Solupore technology on T cellfunctionality, the capacity of transfected PBMC to induce GvHD in a NSGmouse model was studied. NOD scid gamma mice (NSG mice) is anart-recognized immunodeficient laboratory mouse strain from The JacksonLaboratory.)

If transfected cells were adversely affected by the Solupore deliverytechnology, their ability to engraft and induce Graft versus HostDisease (GvHD) would be impaired. A comparison with nucleofection wasalso carried out. It was impractical to include Neon electroporation asa comparator because the high loss of cells in the process would requirean unfeasibly high number of cells to be electroporated.

Method: 3 kDa Dextran-Alexa488 was delivered to 20×10⁶ and 5×10⁶ PBMC bysoluporation and nucleofection respectively. Soluporation was carriedout using the 44.45 mm Stirred Cell system. A monolayer of cells wasformed on a 1 μm PCTE hydrophilic membrane (Sterlitech) by applying apressure of 100 mbar for 15-25 secs until all media was removed. 5 ml ofdelivery solution containing 3 μM Dextran 3000 was prepared and loadedinto the LP100 atomiser. Cells were soluporated and after 1 min and 30sec, the membrane was gently transferred to a 60-mm cell culture gradepetri dish. After 2 mins, 1 ml of stop solution was added to themembrane and left to incubate for 30 seconds, after which time, 4 ml ofmedia was added to the cells. The petri dish was transferred to anincubator at 37° C. with 5% CO² for 30 mins. For nucleofection, 5×10⁶cells were used per transfection. Cells were harvested, washed twice in1× PBS and resuspended according the weight of the mouse i.e. 1×10⁶ pergram. injected intravenously via the tail vein based on weight per mouse(1×10⁶ per gram). Mice were weighed 2-3 times weekly and monitored forthe appearance of GvHD-like symptoms. Peripheral blood was collectedbetween 9-12 days post-injection and processed for flow cytometryanalysis. Similarly, blood and spleen were collected at the end of thestudy and prepared for analysis.

PBMC were isolated from three different donors (D119, D120, D121) andcells from each donor were soluporated or nucleofected. There were fourgroups of NOD scid gamma (NSG) mice, with 5 mice per group, for thestudy. The groups were: 1. No cells 2. UT 3. Soluporation 4.Nucleofection (4 groups×5 mice p/group×3 donors=60 mice). Cells wereinjected into the mice on Day 0. Animals were monitored daily and GvHDsymptoms were present by Day 14 post injection in all animals thatreceived cells indicating that cells were viable and functional. Bloodsamples were taken from the mice on Day 14 and analysed by flowcytometry for the presence of human PBMC, as indicated by CD45expression, and T cell subsets. Results demonstrated that CD45+ cellswere present in the untreated groups and soluporated groups at thistimepoint, confirming that the cells were viable and functional (FIGS.53A, B, C). Lower numbers of CD45+ cells were detected in nucleofectedcells at this timepoint. These data demonstrate that soluporated humanPBMC remain viable and functional in vivo such that they are capable ofinducing GvHD in NSG mice. The results demonstrate successfulengraftment of soluporated cells into the NSG mice indicating that theirviability and functionality is retained following soluporation.

Filter Membrane Conditions for Delivery of Payload Composition to Cells

To facilitate and to enhance the exposure of cells (non-adherent cellsor adherent cells) to permeableisation solution, the filter membrane isoptionally vibrated before or after or during delivery or permutationsof these. To assist in the formation of a monolayer of cells on a filtermembrane, the membrane can vibrate before or after or during delivery orpermutations of these. Vibration of a filter membrane can be carried outusing a number of readily available devices, e.g., PiezoelectricAccelerometers such as a Miniature Triaxial DeltaTron® Accelerometers(available from Bruel and Kjaer, www.bksv.com). A number of othersuitable vibrating motors are also available from Precision Microdrives;www.precisionmicrodrives.com.)

For example, the vibration may be brought about by an eccentric rotatingmass (ERM) system or a linear resonant actuator (LRA) system. Bypreference, 1, 2 or 3 actuators (LRA) corresponding to the X, Y and Zaxis may be attached to the membrane or membrane holder such that themembrane vibrates by mechanical coupling to the actuator.

The advantage of the LRA system is that each axis may be drivenindependently. Accordingly, complex but controllable vibration patternsmay be developed on the membrane. Additionally, identification ofmechanical resonance points due to the physical character of themembrane will improve the degree of control that may be exhibited overthe membrane. A 3 axes accelerometer device will be mechanically coupledto the filter membrane or holder to feedback the excursions experiencedby the membrane. The Accelerometer system may be used to monitor or as acontrol feedback signal to the vibrational system, generating an errorsignal between the desired vibrational pattern and the achievedvibrational pattern. The selection of driving vibrational frequencies ismade based on the stiffness of the membrane and the size of cells on themembrane. An example vibrational pattern is brought about withsinusoidal signals at 3000 Hz on the x and y axes and no signal on the zaxis. The excursions are 1 mm peak to peak and the x and y drivingwaveforms are coherent with no phase difference between them. Many otherpatterns are possible including ones that lead to swirling, shaking inthe x, y and/or z axes.

Device for Delivering Cargo Molecule(s) to Mammalian Cells

The current subject matter generally relates the delivery biologicalpayloads to cells. The delivery of biological payloads to cells caninvolve the atomization and delivery of a permeabilizing solution onto amonolayer of cells. Current techniques can fall short in a number ofareas, which is discussed in more detail below.

Prior to receiving the payload, the cells can be submersed or suspendedwithin a culture medium. To achieve effective payload uptake, it can bebeneficial to remove the culture medium so that cells can be exposed tothe permeabilizing solution, and to organize the cells in a monolayerconformation. With certain current techniques, the medium is removed bycentrifugation. Although this technique can be effective at removing themedium, it typically does not result in the formation of a uniformmonolayer of cells.

In order to ensure an appropriate distribution of the permeabilizingsolution on the monolayer of cells, the cells can be aligned under asolution atomizer or nebulizer. With current techniques, cells aregenerally aligned under the atomizer using a manual system, which issubject to operator variability. The atomizer/nebulizer can be used todispense the permeabilizing solution onto the monolayer of cells.However, certain atomizers are designed to dispense volumes of solutionon the order of magnitude of milliliters, while dispensation volumes onthe order of microliters can be preferable, or even required.Additionally, the transfection protocol for payload delivery can involveseveral time critical steps. Currently, handling of fluids is generallycontrolled manually, and it is therefore intrinsically variable.

Because of these shortcomings, the data generated can be inherentlyinconsistent. The lack of reproducibility of the data can hamper furtherdevelopment of the payload delivery process. In order to address theaforementioned issues, some aspects of the current subject matterprovides a delivery system that enables greater consistency in thedelivery process, and higher efficiency of delivery, while maintainingcell health.

Example Delivery System

FIG. 65 shows an example of a delivery system 8800 configured to delivera payload to cells. The delivery system 8800 can include a housing 8802configured to receive a plate 8804 comprising a well. The deliverysystem 8800 can include a differential pressure applicator 8806configured to apply a differential pressure to the well, a deliverysolution applicator 8808 configured to deliver atomized deliverysolution to the well, a stop solution applicator 8810 configured todeliver a stop solution to the well, and a culture medium applicator8812 configured to deliver a culture medium to the well.

As an example, in some embodiments, the differential pressure applicator8806 can be, or can include, a nozzle valve assembly, e.g., the nozzlevalve assembly 9310, described below with regard to FIGS. 84, and 89-91.As another example, the differential pressure applicator 8806 can be, orcan include, a vacuum manifold assembly, e.g. the vacuum manifoldassembly 9008, described below with regard to FIGS. 93-107. In someembodiments, the delivery solution applicator 8808 can be, or caninclude a nebulizer such as, e.g., nebulizers 9304, 9804 described belowwith regard to FIGS. 85, 88, 93, and 115-118. In some embodiments, thestop solution applicator 8810 can be, or can include needle emitterssuch as, e.g., needle emitters 9303 described below with regard to FIGS.85, 87. As another example, in some embodiments, the culture mediumapplicator 8812 can be, or can include, the needle emitters 9303.

The delivery system 8800 can also include a control system 8814, anactuation system 8816, a support frame 8818, and a sensing andmanagement system 8820. In some embodiments, the differential pressureapplicator 8806, delivery solution applicator 8808, stop solutionapplicator 8810, and/or the culture medium applicator 8812 can becoupled to the support frame 8818. The actuation system 8816 can coupledto the support frame 8818, the housing 8802, and/or the plate 8804, andcan be configured to move the support frame 8818, the housing 8802,and/or the plate 8804. As another example, the actuation system 8816 canbe coupled to, and configured to move, the differential pressureapplicator 8806, delivery solution applicator 8808, stop solutionapplicator 8810, and/or the culture medium applicator 8812. For example,the support frame 8818 can be, or can include, the fluidic head module9308, described below with regard to FIGS. 84-89. The actuation systemcan be, or can include, actuator 9319, described below with regard toFIGS. 84, 85. In some implementations, a vibration system can beincluded to vibrate a membrane (e.g., located within the well of theplate).

The sensing and management system 8820 can include sensors and/orthermal management systems. As an example, the sensors and/or thermalmanagement system can be coupled to the differential pressure applicator8806, delivery solution applicator 8808, stop solution applicator 8810,and/or the culture medium applicator 8812, and can be configured tomeasure and/or control pressures, temperatures, positions, and flowrates.

The control system 8814 can include at least one data processor and canbe electrically coupled to the actuation system 8816 and the sensing andmanagement system 8820. The control system can be configured to controlthe actuation system 8816, as well as the sensing and management system8820. As an example, the control system 8814 can be, or can include, thecontrol system 9306 described below with regard to FIG. 84.

Example Vacuum Pressure System

FIG. 66 shows a diagram 8900 that illustrates nine elements of adelivery system. The system includes: an addressable well vacuummanifold assembly; atomization; fluid control; temperature control of adelivery solution; mounting; automation and software; enclosure;filters; and temperature control of a base plate.

The delivery system can address the following areas of variabilityrelated delivering a payload to cells: monolayer formation; atomization;automation of the payload delivery; and temperature control of thesolutions and the culture container.

In order to improve the consistency of the data and the deliveryefficiency of payload delivery, the system removes certain known sourcesof variability from the process. The system can address: removal ofmedia and creation of a monolayer of cells using a vacuum; atomizationof the permeabilizing solution to produce monodispersed droplets;fluidic control of the solutions to enable automation; temperaturecontrol of the solution; mounting of a spray head and a temperaturereservoir; automation and software design; enclosure for the instrument;and temperature control of a base plate.

FIGS. 67-69 show an exemplary embodiment of a precision rig system 9000.The precision rig system 9000 can include needle emitters 9002, anatomizer 9004, and a vacuum manifold system 9006. The vacuum manifoldsystem 9006 can include a vacuum manifold assembly 9008, a translationalstage 9010, valves 9011 (shown in FIGS. 68-69), and a manifold 9024. Insome embodiments, the vacuum manifold assembly 9008 can be coupled to atranslational stage 9010 via coupling members 9012. In some embodiments,the valves 9011 can be pinch valves. The vacuum manifold assembly 9008can include a filter plate 9014, a base plate 9016, and a top plate 9018(shown in FIGS. 68-69). As shown in FIGS. 68-69 the filter plate can bea 96-well filter plate. The filter plate 9014 can seat within a recessedregion of the base plate 9016, and the top plate can be positioned overthe filter plate 9014 and coupled to the base plate 9016 to secure thefilter plate 9014 in position.

The needle emitters 9002 can function to deliver a culture medium, whichcan contain cells, to wells of the filter plate 9014. The base plate9016 can have vacuum couplings 9020 extending from a bottom surfacethereof. The vacuum couplings 9020 can generally be in the form ofcylindrical tubes, and can allow vacuum pressure to be applied tocorresponding wells of the filter plate 9014. Vacuum pressure can berouted through ports 9024 a of the manifold 9024 to each valve 9011, andto the vacuum couplings 9020. The atomizer 9004 can atomize apermeabilizing solution and deliver it to cells within a well of thefilter plate 9014. Systems, devices, and methods related to the deliverya permeabilizing solution onto a monolayer of cells are discussed inmore detail below.

As shown in FIGS. 68-69, the precision rig system 9000 can include guiderail 9022 that can extend along an X axis. In some embodiments, thetranslational stage 9010 can be coupled to a guide rail 9022, which canallow the vacuum manifold assembly 9008 to be translated along the Xaxis. This can allow the vacuum manifold assembly 9008 to be movedrelative to other components such as, e.g., the needle emitters 9002and/or the atomizer 9004.

FIGS. 70-71 show exploded views of the vacuum manifold assembly 9008.FIGS. 72-73 show a top view and a side cross-sectional view of the baseplate 9016, respectively. As shown in FIGS. 70-71, the vacuum manifoldassembly can include the filter plate 9014, the base plate 9016, gaskets9026, and the top plate 9018 which can have coupling bores 9018 b.

Referring to FIGS. 70-73, the base plate 9016 can include first andsecond sets coupling bores 9016 b, 9016 c. The first set of couplingbores 9016 b can align with coupling bores 9018 b in the top plate 9018such that the base plate 9016 can be coupled to the top plate 9018 via acoupling element such as, e.g., a bolt or screw, that can extend throughthe coupling bores 9016 b, 9018 b. The coupling members 9012 can becoupled to the base plate 9016 via coupling elements that can extendinto the second set of coupling 9016 c.

The base plate 9016 can include a first recessed region 9028 where thefilter plate 9014 can be received, or seated, as well as secondaryrecessed regions 9030 that can receive gaskets 9026. Each of thesecondary recessed regions can have openings 9030 b or passages that cancouple with corresponding vacuum couplings 9020.

The filter plate 9014 can have wells that have active openings 9014 b,in addition to having wells that have inactive openings 9014 c. Theactive openings 9014 b can be positioned over, and fluidly coupled to,openings 9030 b in the base plate 9016. In other words, certain wells ofthe filter plate 9014 can be active, while other wells of the filterplate 9014 can be inactive.

The gaskets 9026 can have bores 9026 b that can align with activeopenings 9014 b of wells in the filter plate 9014 and with the openings9030 b in the base plate 9016. The gaskets 9026 can function to formseals between the base plate 9016 and the filter plate 9014, therebyisolating each active opening 9014 b from other active openings 9014 b,as well as from inactive openings 9014 c, while allowing fluidcommunication between the active openings 9014 b of the wells andcorresponding vacuum couplings 9020.

FIGS. 74-76 show various view of the top plate 9018. As shown in FIGS.70-71, and 9-11, the top plate 9018 can include a recessed region 9032that can receive a portion of the filter plate 9014. When the vacuummanifold assembly 9008 is assembled, the gaskets 9026 can be seated inthe secondary recessed regions 9030 of the base plate 9016, the filterplate 9014 can be received within the first recessed region 9028, andthe top plate can be positioned over the filter plate 9014 and coupledto the base plate 9016 as described above.

In some embodiments, all of the wells of the filter plate 9014 can becoupled to different valves 9011, and to a manifold, such that all ofthe wells can be active.

Removal of Media and Creation of a Cell Monolayer Using a Vacuum

The vacuum manifold system 9006 can remove a culture medium from between1 and 12 wells of the filter plate 9014, with a total of 6 wells beingaddressable at a one time. FIGS. 77-78 show top views of the filterplate 9014. In FIG. 77 the filter plate 9014 is in a first position suchthat wells F2, F4, F6, F8, F10, and F12 can be active when the filterplate 9014 is received within the first recessed region 9028 of the baseplate 9016. The filter plate 9014 can be rotated 180° such that wellsC1, C3, C5, C7, C9, and C11 are active, as shown in FIG. 78.

As described above, vacuum pressure can be routed through a manifold9024 to each valve 9011, and to the vacuum couplings 9020. FIGS. 79-81show various views of a portion of the precision rig system 9000. Asshown in FIGS. 79-81, the manifold can include 8 ports 9024 a, andtubing 9034 can extend from 6 ports 9024 a on the manifold to ports onthe 6 valves 9011. The tubing can also connect a port on each valve 9011to the vacuum couplings 9020 connected to the base plate 9016. A vacuumline (not shown) can be connected to 1 of the 2 remaining open ports9024 a on the manifold 9024, and the remaining port 9024a can be sealed.

The vacuum manifold system 9006 can be used to enable formation of amonolayer of suspension cells. As described above, needle emitters 9002can deliver a culture medium, which can contain cells, to wells of thefilter plate 9014. Vacuum pressure can be applied to the 8-channelmanifold via the vacuum line. The valves 9011 can be opened or closedindividually. Therefore, access to each active well can be controlledindividually. When the valves 9011 are open, the applied vacuum pressurecan be sufficient for effective removal of the culture medium in whichthe cells are suspended, without causing shear force that can lead todamage. The extracted medium can travel through the tubing 9034 and thevalves 9011, through the manifold 9024, and out of the port 9024a wherethe vacuum line is attached. In this way, a cell monolayer can be formedand cell viability can be maintained. The formation of the cellmonolayer can be achieved without damaging cells.

The vacuum manifold system 9006 has been built and tested. Duringtesting a culture medium having cells was added to wells of a 96-wellfilter plate 9014. Vacuum pressure was applied via the manifold 9024.Vacuum pressure ranging from 10 bar to 100 mbar was applied toindividual wells by opening the well's associated valve 9011.

At higher vacuum pressures, between −100 mBar and −75 mBar, the culturemedium was removed far enough away from the well that when the filterplate 9014 was removed, the culture medium did not “wick” back into thewell.

At lower pressures, between −50 to −10 mBar, the media stayed closeenough to the well that when the filter plate 9014 was removed from thevacuum manifold assembly 9008, some of the medium “wicked” back in tothe well. The amount of liquid that wicked back into the well wasbetween approximately ˜5-10 μl. This may not be a problem when thevacuum manifold assembly 9008 is part of the precision rig system 9000as the well will be sprayed and have the culture medium replaced beforethe filter plate 9014 is removed from the manifold.

In some embodiments, filter paper, or other materials, can be used toprevent wicking. For example, filter paper can be placed between thegaskets 9026 and the base plate 9016 to prevent wicking of the mediaback into the well.

At pressures, as low as −10 mBar the culture medium was removed at asedate, controlled rate which was gentler on the cells. This occurredboth in presence and absence of cells. This can lead to better viabilityand recoverability of cells from the wells.

During testing, pulsing the valves 9011 on and off did not appear to aidin the removal of the culture medium.

Centrifugation was also investigated as a method of forming a cellmonolayer. However, this was found to be ineffective as it formed anuneven layer of cells. FIG. 82 shows a distribution of GlowGermparticles that were observed following centrifugation and vacuumextraction. GlowGerm particles include. The top 3 images show thedistribution of GlowGerm particles that resulted from using vacuumpressure to remove the culture medium, and the bottom 3 images show thedistribution of GlowGerm particles that resulted from usingcentrifugation to remove the culture medium. The centrifuged sampleswere centrifuged at 350×g for 5 min, and the vacuum pressure sampleswere vacuumed for 15 sec at −800 mbar.

As shown in FIG. 82, the vacuum pressure samples show a more evendistribution of GlowGerm particles, whereas an uneven, crescent shapeddistribution of GlowGerm particles was observed followingcentrifugation.

Comparable results of mRNA uptake using vacuum and spin methods wereachieved, even at −10 mBar. The GFP MFI was found to be greater insamples prepared by the vacuum vs spin (˜300,000 vs 50,000 units). Thisindicates that more mRNA got into the positive cells.

The time needed to extract the culture medium from the wells isestimated to be between approximately 3 s and 20 s. However, the amountof time that it takes to remove the culture medium from the wells can bedependent on the amount of vacuum pressure that is applied. More workcan be done to test the length of time required to remove the media atthe lower vacuum pressures.

In some implementations, it is possible to be able to evacuate all wellsat the same time at a lower pressure to remove residual media from thespouts (no plate on the manifold).]

The vacuum manifold system 9006 allows vacuum pressure to be applied toindividual wells of a filter plate 9014. By applying a vacuum pressureto individual wells on a filter plate 9014, greater precision control ofthe vacuum pressure, and greater consistency of the vacuum pressureapplied to each well, can be achieved. Existing vacuum manifolds apply avacuum to an entire 96-well filter plate. During testing, lower vacuumpressures were effective in removal of the culture medium and creationof an even monolayer of cells within the well.

Example Positive Pressure System

FIG. 83 shows a diagram 9200 that illustrates six elements of an examplepositive pressure delivery system. The elements include: atomization;fluid control; positive pressure; a modular head; automation andsoftware; and an enclosure.

The positive pressure delivery system can address the following areas ofvariability related delivering a payload to cells: monolayer formation;atomization; automation of the payload delivery; and temperature controlof the solutions and the culture container.

In order to improve the consistency of the data and the deliveryefficiency of payload delivery, the system removes certain known sourcesof variability from the process. The system can address: removal ofculture medium and creation of a monolayer of cells using a positivepressure; atomization of the permeabilizing solution to producemonodispersed droplets; fluidic control of the solutions to enableautomation; temperature control of the solution; mounting of a emitters,atomizers, nebulizers, and a temperature reservoir; automation andsoftware design; enclosure for the instrument; and temperature controlof a base plate configured to retain a well plate.

FIGS. 84-85 shows an exemplary embodiment of a positive pressure system9300. This positive pressure system 9300 utilizes positive pressure asan alternative to vacuum for removal of the culture medium. Positivepressure provides greater accuracy and precision of delivery of lowvolumes (1 μl-100 μl) of fluid to force the culture medium from wells ofa well plate. The positive pressure system 9300 can include a mountingarray 9302, a manifold assembly 9305, and an actuation and controlsystem 9306.

The manifold assembly 9305 can include a base 9312, also referred to asa plate holder, that can extend from a housing 9314 of the manifoldassembly 9305. The base 9312 can be configured to receive filter plate9316. As shown in the illustrated example, the filter plate 9316 can bea 96-well filter plate.

The mounting array 9302 can include fluidic head modules 9308 havingneedle emitters 9303, nebulizers 9304, and/or positive pressure nozzleassemblies 9310 attached thereto. The needle emitters 9303 can beconfigured to deliver a culture medium, which can contain cells, and astop solution to wells of the filter plate 9316. The nebulizers 9304 canbe configured to atomize a permeabilizing solution and deliver it tocells within wells of the filter plate 9316. The positive pressurenozzle assembly 9310 can be configured to apply a positive pressure towells of the well filter plate 9316 to remove a liquid portion of theculture medium from the well, thereby creating a monolayer of cells.Systems, devices, and methods related to the delivery a permeabilizingsolution onto a monolayer of cells are discussed in more detail below.

In some embodiments, the positive pressure system 9300 can include aguide rail 9318. In the illustrated example, the filter plate 9316 canbe held stationary, and a position of the mounting array 9302 can beadjusted relative to the position of the filter plate 9316. For example,the mounting array 9302 can be coupled to a guide rail 9318, which canallow the mounting array 9302 to be translated along an axis X2 definedby the guide rail 9318. For example, an actuator 9319 can move themounting array 9302 along the guide rail 9318. The guide rail 9318 canalso be moved along an axis Y2, which can be perpendicular to the axisX2. The actuator 9319 can also move the mounting array 9302 along theaxis Y2. Therefore, the mounting array 9302, including the needleemitters 9303, nebulizers 9304, and/or positive pressure nozzleassemblies 9310 can be selectively positioned above wells of the filterplate 9316 when the filter plate 9316 is positioned within the housing9314.

As an example, a user can insert the filter plate 9316 into the base9312, which can then be positioned within the housing 9314. The filterplate 9316 can be held stationary after it is received within thehousing 9314. The filter plate 9316 can be selectively addressed byneedle emitters 9303, nebulizers 9304, and/or positive pressure nozzleassemblies 9310 coupled to fluidic head modules 9308 of the mountingarray 9302. In the illustrated example, the mounting array 9302 includessix fluidic head modules 9308. Two fluidic head modules 9308 includeneedle emitters 9303, three fluidic head modules 9308 include nebulizers9304, and one fluidic head module 9308 includes a positive pressurenozzle assembly 9310. The mounting array 9302 can be moved along theaxes X2, Y2, such that each of the fluidic head modules 9308, includingthe needle emitters 9303, nebulizers 9304, and/or positive pressurenozzle assemblies 9310, can address any location on the filter plate9316.

In some embodiments, each of the individual fluidic head modules 9308can have the capability to move up and down parallel to an axis Z2,which allows for independent actuation of the fluidic head modules 9308.In some embodiments, movement of the fluidic head modules 9308 andactuation of needle emitters 9303, nebulizers 9304, and/or positivepressure nozzle assemblies 9310, are controlled independently. Themovement of one or more of the fluidic head modules 9308, and actuationof needle emitters 9303, nebulizers 9304, and/or positive pressurenozzle assemblies 9310 attached thereto, can occur contemporaneously. Insome implementations, the fluidic head modules 9308 activate the needleemitters 9303, nebulizers 9304, and/or positive pressure nozzleassemblies 9310 when they move toward the filter plate 9316. The fluidichead modules 9308 can accommodate a variety of fluid dispensingassemblies, including but not limited to, needle assemblies, Ari Mistnebulizers, and positive pressure nozzle assemblies, as shown in FIGS.84-85.

The actuation and control system (ACS) 9306 can include at least onedata processor, and can control movement of the mounting array 9302and/or the fluidic head modules 9308. The ACS 9306 can also controlactuation of the needle emitters 9303, nebulizers 9304, and/or positivepressure nozzle assemblies 9310 attached to the fluidic head modules9308. For example, the ACS 9306 can include software, havinginstructions that can be interpreted by the data processor of the ACS9306. The data processor can receive the instructions, process theinstructions, and execute the instructions. For example, the dataprocessor can deliver control signals to actuators and/or motors of theACS 9306 to adjust a position of the mounting array 9302, and/or toactuate needle emitters 9303, nebulizers 9304, and/or positive pressurenozzle assemblies 9310.

Example Fluidic Head Module

As described above, the fluidic head modules 9308 provide mountingpoints for various fluidic dispensing assemblies (e.g., the needleemitters 9303, nebulizers 9304, and/or positive pressure nozzleassemblies 9310) such that they can be coupled to the mounting array9302. The fluidic head modules 9308 also provide actuation along theaxis Z2 such that the dispensing assemblies can be selectively actuated.

FIG. 86 shows an enlarged view of a fluidic head module 9308. Thefluidic head module 9308 can have a frame 9350 that is includes a seriesof machined aluminum pieces, which create the frame 9350. For example,the frame 9350 can include a base plate 9352, a back plate 9354, andupper and lower mounting elements 9356, 9358. The machined aluminumpieces can allow for high tolerance locating of the fluidic head module9308.

The fluidic head module 9308 can include a shaft 9360 that extendsbetween the base plate 9352 and the upper mounting element 9356. Theshaft 9360 can extend through an opening in the lower mounting element9358. A guide 9362 such as, e.g., a ball spline, can be mounted on theshaft 9360 and coupled to the lower mounting element 9358. The guide9362 facilitates vertical motion of the fluidic dispensing assemblies(e.g. the needle emitters 9303, nebulizers 9304, and/or positivepressure nozzle assemblies 9310) along axis Z2. For example, a pneumaticactuator 9364 positioned between the upper and lower mounting elements9356, 9358 can drive the lower mounting element 9358 up and down alongthe shaft 9360. The pneumatic actuator 9364 can drive associatedpneumatic fittings as well as two proximity sensors. For example, thesensors can be embedded in a wall of the cylinders. FIGS. 87, 88, and 89the fluidic head module 9308 with a needle emitter 9303, a nebulizerassembly 9301 including a nebulizer 9304, and a positive pressure nozzleassemblies 9310 mounted thereon, respectively.

Removal of Media and Creation of a Cell Monolayer Using PositivePressure

As described above, positive pressure nozzles can be configured to applya positive pressure to wells of a well filter plate to remove a culturemedium and create a monolayer of cells. FIGS. 90-91 show magnified viewsof the positive pressure nozzle assembly 9310 used with the positivepressure system 9300 shown in FIGS. 84-85. In the illustrated example,the nozzle assembly 9310 includes an internal lumen, which can be formedfrom a tube 9320, positioned within a housing 9322. The tube 9320 canhave openings 9321, 9323 adjacent first and second ends 9326, 9327 ofthe positive pressure nozzle assembly 9310. In some embodiments, thehousing 9322 can provide structural stability for the tube 9320.

As shown in FIG. 90, the nozzle assembly 9310 includes a couplingelement 9324 coupled to a first end 9326 of the tube 9320. The couplingelement 9324 can be configured to form a seal with the opening 9321adjacent to the first end 9326 of the tube 9320 such that pressurizedvapor (e.g., air) can be delivered to a well 9316 a of the filter plate9316. For example, compressed air can be delivered to an internal lumen9325 of the coupling element 9324 from a compressor. The internal lumen9325 of the coupling element 9324 can be in fluid communication with thelumen of the tube 9320 via the opening 9321, the lumen of the tube 9320can be in fluid communication with the well 9316 a via the opening 9323.The compressed air can increase a pressure within the tube 9320, therebyincreasing pressure within the well 9316 a. Accordingly, the positivepressure nozzle assembly 9310 controls the flow and pressure ofcompressed air.

As shown in FIG. 91, the second end 9327 of the tube 9320 includes asealing element 9328 positioned adjacent to the opening 9323, outside ofthe tube 9320. The sealing element 9328 is configured to encompass thewell 9316 a and form a seal with an upper surface 9330 of the filterplate 9316, thereby isolating the well 9316 a. The sealing element 9328is configured to prevent air from leaking between the second end 9327 ofthe nozzle assembly 9310 and the upper surface 9330 of the filter plate9316, thereby improving accuracy and precision of control of thepressure within the well 9316 a. For example, when the nozzle assembly9310 is engaged with the filter plate 9316, the application ofcompressed air will force liquid to flow through a filter base 9332 ofthe individual well.

In some embodiments, a positive pressure nozzle assembly can include avalve positioned along its length. The valve can function as a manualcontrol to control a pressure of air delivered to a well of a filterplate. FIG. 92 shows an example of a portion of a nozzle assembly 9410that includes a valve 9432. In the illustrated example, the nozzleassembly 9410 includes a first tube portion 9420 a and a second tubeportion 9420 b that are coupled to the valve 9432. The second tubeportion 9420 b can include a sealing element positioned adjacent to adistal opening 9423 of the second tube portion 9420 b. The nozzle cangenerally function similarly to the nozzle assembly 9310, describedabove with regard to FIGS. 90-91. For example, the distal end of thesecond tube portion can be positioned adjacent to an upper surface of afilter plate such that the sealing element 9428 encompasses an openingof the well, and forms a seal with the upper surface of the filterplate. Compressed air can be delivered to an internal lumen of the firsttube portion 9420 a. When the valve is in an open position, the air cantravel through the valve 9432, through the second tube portion 9420 b,and into the well of the filter plate. Alternatively, the valve 9432 canbe adjusted to an off position to prevent air flow to the well. Thevalve 9432 can be adjusted to control air pressure within the well.

Atomization of the Delivery Solution to Produce Monodispersed Droplets

FIG. 93 shown an enlarged view of the nebulizer assembly 9301 that canbe coupled to a fluidic head module 9308. In the illustrated example,the nebulizer assembly 9301 includes a syringe 9366, a micro valve 9368,and a nebulizer 9304. The nebulizer 9304 can be coupled to the microvalve 9368 via a coupling element 9370 (e.g., a precolumn coupler). Themicro valve 9368 can be retained within, and/or coupled to, a valveholder 9372, which can be coupled to the syringe 9366 via an adapter9374. The nebulizer assembly 9301 enables high accuracy and precision ofdelivery of payload solutions to the nebulizer 9304.

There are a number of different ways in which a permeabilizing solutioncan be delivered onto a monolayer of cells. For example, thepermeabilizing solution can be atomized using ultrasonication or it canbe nebulized using a nebulizer.

Both ultrasonication and nebulization were tested as delivery methods. Atotal of 4 different spray heads were tested.

The following parameters were assessed for each spray head: Airpressure, flow rate, distance, volume delivered, cell number, time ofspray, frequency of ultrasonic probe, and power of ultrasonic probe.

The effect on the character of the spray was assessed using high-speedcamera recording. The force experienced by the cells was determined byforce sensor analysis. The volume delivered into the well was assessedusing a colorimetric assay.

Ultrasonication tests were performed at 60 kHz, 130 kHz, and 180 kHz.Liquid can be driven to an ultrasonic nozzle by a pumping system, and itcan be atomized into a fine mist spray using high frequency soundvibrations.

Piezoelectric transducers were used to electrical input into mechanicalenergy in the form of vibrations, which created capillary waves in theliquid when introduced into the nozzle, and resulted in atomization ofthe liquid. Each ultrasonic probe worked at a given resonant frequency.The operating frequency can determine the size of the liquid dropletsgenerated. The size of the droplets can also be affected to a lesserextent by the power at which the ultrasonic probe is operated. Anancillary air stream can be used to help control and shape the spray.

Nebulization tests were performed using an Ari Mist nebulizer 9304 (FIG.125). As shown in FIG. 125, the nebulizer 9304 includes a connection9307 for liquid and a connection 9309 for air. The Ari Mist nebulizeroperates on compressed gases and requires a pump to supply the samplesolution. This atomizer has two channels, one for the gas (air) and theother one for the liquid to be nebulized, which run along parallelpaths. Both paths end at the tip of the nebulizer with an orifice forthe gas and an exit for the liquid. The gas flow can draw the liquidinto the gas stream. The impact with the gas molecules can break theliquid into small droplets, resulting in nebulization.

The ultrasonic emitter operating at 180 kHz proved to be more effectivecompared to the 130 kHz, 60 kHz ultrasonic heads and the Ari Mistnebuliser in delivering payloads to T-cells. FIG. 94 shows a series ofresults characterizing the efficiency of payload delivery for theultrasonic emitter operating at 180 kHz. The test results indicated thatpayloads had been delivered to T-cells successfully, at an efficiency ofapproximately 15-25%, with high level of consistency between replicates(±1%). The health of the cells was maintained following delivery (85%viability).

The results indicate that the ultrasonic spray emitter generates amonodispersed spray which results in even deposition of the deliverysolution and payload onto cells. Additionally reducing the volumedelivered and reducing the ethanol concentration improved deliveryefficiency with the ultrasonic spray head.

FIG. 95 shows another series of results characterizing GFP uptake forthe ultrasonic emitter, the Ari Mist nebulizer, and a MAD nasal sprayemitter. The MAD Nasal™ Intranasal Mucosal Atomization Device (Teleflex3015 Carrington Mill Blvd, Morrisville, N.C. 27560) was used to atomizedelivery solution containing payload. Briefly, 7 μl of delivery solutionwas pipetted directly into the nasal head. The spray head was directlyconnected to an air pressure source via a luer lock connection. 1.5 barair pressure was supplied to the spray head over 330 ms to generate thespray. The ultrasonic spray emitter operating at 180 kHz is moreefficient at delivering mRNA to T cells based on current data. A 32.7%GFP uptake was obtained with the ultrasonic spray emitter compared to24.8% with the Ari Mist nebulizer (e.g., nebulizer 9304). Bothultrasonic emitter and Ari Mist nebulizer resulted in higher GFP mRNAdelivery compared to the NAD nasal spray emitter which resulted in 16.9%delivery efficiency.

FIG. 96 shows a results characterizing cell viability for the ultrasonicemitter, the Ari Mist nebulizer, and the MAD nasal spray emitter. Theresults represent a minimum of three technical repeats for each sprayhead (e.g., the ultrasonic emitter, the Ari Mist nebulizer, and the MADnasal spray emitter) that was tested. The Ari Mist nebulizer resulted inbetter cell viability at 85.3% relative to untreated cells, compared tothe ultrasonic head which had an average of 72.3% cell viabilityrelative to untreated cells. The MAD nasal spray emitter resulted in thehighest cell viability with 99.7% cell viability relative to untreatedcells.

Enclosed Atomization

During delivery of a solution using e.g., an atomizer, nebulizer, and/orultrasonic emitter, a fine aerosol is generated. In some cases, the fineaerosol may contaminate adjacent wells of a multi-well filter plate. Insome embodiments, a distal end portion of an atomizer, nebulizer, and/orultrasonic emitter can be enclosed, which may prevent contamination ofadjacent wells of the filter plate.

Three rig platforms were built: rig 1 (R1), rig 3 (R3) and rig 4 (R4)(FIGS. 126-128). Table below shows several feature of the rigs.

As illustrated in FIG. 126, R1 11700 includes a solution reservoir 9810(e.g., an Elveflow sample reservoir) configured to provide apermeabilizing solution to an Ari Mist nebulizer 9804, and a pinch valve9808 (e.g., an Elveflow pinch valve) configured to control delivery ofthe permeabilizing solution to the nebulizer 9804. The nebulizer 9304can be mounted on a spray head mount 11714 which can be configured toretain the nebulizer 9304 and facilitate alignment of the spray head.The pinch valve 9808 is configured to enable fluidic control of apayload solution to the nebulizer 9304. The spray head mount 11714 canbe mounted on a guide 11717 configured to facilitate vertical motion ofthe spray head mount 11714 along a vertical axis Z3. The spray headmount 11714, including the nebulizer 9804, can be mounted above a plateholder 11718 that can be configured to receive a filter plate 11716.

As illustrated in FIG. 127, R3 11800 includes a nebulizer assembly 9301having a sample reservoir syringe 9366 coupled to an Ari Mist nebulizer9304 via a micro valve 9368 configured to control delivery of apermeabilizing solution to the nebulizer 9304. The nebulizer assembly9301 can be mounted to a fluidic head module 9308. The fluidic headmodule 9308 can be mounted on a guide 11717 configured to facilitatevertical motion of the fluidic head module 9308 along a vertical axis.

As illustrated in FIG. 128, R4 9800 includes a LB-100 atomizer (notshown) coupled to a pinch valve 9808 (e.g., an Elveflow pinch valve)configured to control delivery of the permeabilizing solution from asample reservoir (e.g., Elveflow sample reservoir, not shown) to theatomizer The atomizer can be positioned within a collar 9816 of a sprayhead mount 9814 which can be configured to retain the atomizer andfacilitate alignment of the spray head. The collar 9816, including theatomizer, can be positioned over an opening of a stirred cell system11900 such that the atomizer can deliver a payload to cells within thestirred cell system 11900.

TABLE Features of Rigs. Spray head Valve controlling Sample Spray Headholder payload delivery reservoir Rig 1 Ari Mist Avectas holder Pinchvalve Elveflow (1.5 ml Eppendorf) Rig 3 Ari Mist Spray head Micro valveBD syringe mounted on a fluidic head module Rig 4 LB-100 Avectas holderPinch valve Elveflow (50 ml tube)

Below is a detailed description of the Rig features which enable finecontrol of the spray parameters.

In some embodiments (e.g., R1 and R4), fluidic control of the deliverysolution containing the payload can be achieved using the Elveflow pinchvalve. The fluidic control can be achieved by a fluid control systemthat can apply a constant pressure to an Elveflow fluidic reservoir todrive the fluid through a pinch valve 9808. A volume of fluid that canbe dispensed can be controlled by at least: an amount of pressureapplied; a length of time the valve 9808 is open, and/or a diameter ofthe tubing used. The valve 9808 can be activated by ametal-oxide-semiconductor field-effect transistor (MOS FET) which can becontrolled by a microprocessor.

The Elveflow-Pinch valve 9808 described above had several limitations.For example, re-calibration of the R1 and R4 was required every time thesystem was re-loaded. There was poor accuracy and precision indispensing volumes lower than 5 μl. For low volumes (<5 μl) the relativestandard deviation was approximately 9% over repeated dispenses (10). Toaddress the observed limitations, a R3 was developed. As describedabove, R4 includes the micro-valve 9368 rather than the pinch valve9808. Fluidic control of the delivery solution containing the payloadcan be achieved using the micro valve 9368. R3, which uses the microvalve 9368 had greater accuracy and precision when delivering volumes inthe range of 1 μl to 100 μl.

Fluidic control of air delivered to the nebulizers/atomizers can beachieved using a solenoid valve. In some embodiments, electronics can beused to control actuation of the nebulizers/atomizers. To enableelectronically controlled spray actuation, a system was designed using amicroprocessor based development board to allow easy development of timecontrolled sequences. The development board used the microprocessorPIC16F1619. The spray actuation time and fluid delivery time can bemanipulated through the development board's interface software. Themicroprocessor development board enables pulsing of thenebulizer/atomizer spray. This system was then upgraded to include thehigh speed and repeatable PLC (programmable logic controller) technologyto better align with industry standards and to serve as proof of conceptfor the automated delivery technology (which is based onultra-high-speed PLC technology). The Rig controller consisted of a PLCwith a Gyger controller and a program which facilitates communicationbetween the two pieces of hardware. There is operator interaction to thehardware via a momentary push button.

The Ari Mist nebulizer 9304 parallel path design inherently produces aspray which is off center from a tip of nebulizer 9304. Using a customspray head holder equipped with a goniometer, the alignment of the sprayhead can be adjusted.

Several methods for delivering a solution using an enclosed emitter weretested. The results of GFP uptake using an enclosed emitter werecompared to the results of GFP uptake using an unenclosed emitter.

Method 1: Using a Collar to Enclose the Emitter

FIG. 97 shows an example of a nebulizer assembly 9501 that includes anenclosing collar 9502 positioned around a spray head 9504 of an Ari Mistnebulizer (e.g., nebulizer 9304). In the illustrated example, the sprayhead 9504 is positioned 27 mm above a well 9516 a of a double heightfilter plate 9516. The collar 9502 was designed and manufactured for theAri mist spray head 9504. This collar was installed onto the spray head9504. A 96-well filter plate 9516 with a double height wall was used.

Method 1A: The Collar Forms a Seal with the Well Plate.

At a distance of 26 mm from a tip of the spray head 9504 to a base ofthe well 9516 a, the collar 9502 mated with the top of the well 9516 aof the filter plate 9516 and formed a seal. CD3+ T-cells, 1.5×10⁶, wereseeded in the filter plate 9516 and centrifuged for 5 min at 350×g toremove the culture medium. A delivery solution (4 μl) containing GFPmRNA was sprayed onto the cells and incubated for 2 min. Following the 2min incubation 50 μl of a stop solution was added and incubated for 30s. Finally, 100 μl culture medium was added.

Method 1B: Providing a 1 mm Gap Between the Collar and the Filter Plate

The collar 9502 was installed onto the spray head 9504, and the tip ofthe spray head 9504 was positioned a distance of 27 mm from the base ofthe well 9516 a. Therefore, the collar was held 1 mm above the uppersurface of the filter plate 9516. Note: a single hit protocol (e.g.,single exposure to delivery solution and stop solution) was followed forthe enclosing experiments.

Method 1: Results

FIG. 98 shows results characterizing efficiency (GFP uptake)corresponding to the spray head without the collar 9502, the spray headwith the collar that forms a seal with the filter plate, and the sprayhead with the collar where a 1 mm gap exists between the collar 9502 andthe filter plate.

The tests corresponding to method 1B, where there was a 1 mm gap betweenthe collar 9502 and the upper surface of the filter plate 9516, showsthat the collar 9502 had no impact on GFP uptake. The resultshighlighted comparable data between the wells with and without thecollar 9502 in this set-up. Viability of the T-cells was unaffectedusing the enclosing collar in both set-ups.

Method 1: Conclusions

The result indicate that the collar 9502 does not affect the viabilityof T-cells. The collar 9502 does affect the spray, to varying degrees.This variance may be attributed to the degree to which the system issealed. Sealing the system (e.g., method 1A) led to the formation of alocal pressure maxima which inhibited the spray. Regarding method 1B,providing a gap between the collar 9502 and the upper surface of thefilter plate 9516 provided an outlet for the pressure. The gap allowedfor expansion of the air within the well, and permitted correctactuation of the spray. Therefore, a collar can be used to enclose thespray provided it permits expansion space for the air to expand duringthe spray process.

Method 2: Use of X-Pierce Film to Enclose the Spray.

Rather than enclosing a spray head using a collar, an X-pierce film(Sigma Aldrich, Catalog No. Z722529) was used to enclose the spray. Theuse of an X-pierce film to enclose the spray head may have benefits overthe use of a collar (e.g., collar 9502) for enclosing the spray butavoiding the formation of a seal which can inhibit the spray.

FIG. 99 shows an example of a 96-well PCTE filter plate 9616 with anX-pierce film 9602 adhered to an upper surface of the filter plate 9616.A filter base of the wells of the filter plate 9616 have a 0.4 μm poresize. As shown in the illustrated example, the X-pierce film includes aprecut “X” positioned over opening of each well of the filter plate9616. During the experiment, CD3+ T-cells, 2.5×10⁵, were seeded intoeach well. The filter plate 9616 was centrifuged for 1 min at 350×g toremove the culture medium, thereby creating a monolayer of cells. Theair pressure delivered to the nebulizer was set to 1.65 bar and the tipof the spray head of the nebulizer was positioned 12 mm above an uppersurface of a filter membrane positioned within the well, the filtermembrane having the monolayer of cells formed thereon. A deliverysolution (1 μl) containing GFP mRNA (2 μg) was sprayed onto the cellmonolayer. The cells were then incubated 2 min. Following the 2-minincubation, 50 μl of stop solution was added to each well, and the wellswere incubated for 30 s. 100 μl of culture medium was added to each cellthereafter, as shown in FIG. 99. The filter plate was incubated at 37°C. for 17-24 hours prior to analysis. The single hit protocol wasfollowed for the enclosing experiments.

Method 2: Results

FIG. 100 shows results characterizing efficiency (GFP uptake)corresponding to tests performed with rig 1 (R1) with unenclosed filterplate, tests performed with rig 3 (R3) with an unenclosed filter plate,and tests performed with R3 with an filter plate that included X-piercefilm enclosure over the wells of the filter plate. A description of therigs is provided above. The results indicate that efficiency wascomparable across wells with the film and without the film, therebyindicating the x-pierce film did not have a negative impact on thespray. The use of the X-pierce film did not affect cell viability.

The x-pierce film can be used to enclose the spray and avoid crosscontamination between wells without reducing efficiency (GFP uptake) orcell viability.

Fluidic Control of the Solutions to Enable Automation

In some embodiments, the precision rig can provide an automatedengineering solution for the payload delivery process. To enable this,fine fluidic control of the delivery solution, cold stop solution andculture medium can be used.

The fluidic control can be achieved by fluid control system that canapply a constant pressure to the system to drive the fluid through apinch valve or micro valve. A volume of fluid that can be dispensed canbe controlled by: an amount of pressure applied; a length of time thevalve is open; and/or a diameter of the tubing used.

The valve can be activated by a metal-oxide-semiconductor field-effecttransistor (MOS FET) which can be controlled by a microprocessor.

The fluid control system can allow fluid volumes in the range of 2-10 μlto be delivered using the fine fluidic control. Therefore, the precisionrig system 9000 can control the delivery of metered units of volume inthe range of 2-10 μl. In some implementations, other volumes arepossible.

Temperature Control of the Solutions (Temperature Reservoirs).

In some embodiments, a fluid temperature control system can beimplemented to control the temperature of the delivery solution, stopsolution, and culture medium. The fluid temperature control system caninclude a heating system and a cooling system.

FIG. 101 shows an example of a heating system 11000 that can be usedheat the delivery solution, stop solution, and culture medium. Theheating system can include a fluid reservoir 11002, a housing 11004 anda heating element 11006. The fluid reservoir 11002 can generally be inthe shape of a cylindrical tube that a fluid such as, e.g., the culturemedium can be loaded into. The housing 11004 can be, e.g., an aluminumblock having a passage for receiving the fluid reservoir 11002. Theheating element 11006 can be positioned on a distal end of the housing11004. The heating element 11006 can receive power from a power sourceand can heat the housing 11004, thereby heating the fluid within thefluid reservoir. The heating element 11006 can heat the housing 11004via conduction.

FIGS. 76-77 show an example of a cooling system 11100 that can be usedcool the delivery solution, stop solution, and culture medium. Thecooling system 11100 can include one or more fluid reservoir 11102 suchas, e.g., 1.5 ml Eppendorf tubes, a housing 11104, a cooling element11106 such as, e.g., a thermoelectric cooler, a heat sink 11108, and afan 11110. The delivery and/or stop solutions cab be loaded into the oneor more fluid reservoirs 11102. Power can be delivered to the coolingelement 11106, and the cooling element can generate a hot surface and acold surface. The cold surface can be in contact with the housing 11104,and the hot surface can be in contact with the heat sink 11108. The fan11110 can be coupled to, or positioned over, the heat sink 11108 toremove heat from the hot surface of the cooling element 11106.

In practice, the delivery solution and the stop solution can be held atapproximately 4° C., and the culture medium can be held betweenapproximately 20° C. and 37° C.

Mounting of the Spray Heads and Temperature Reservoirs.

The needle emitters 9002 and the atomizer 9004 can be mounted in asupport. FIG. 104 shows an example of a mounting assembly 11300 that canreleasably retain the needle emitters 9002 and an atomizer 11304. Theatomizer 11304 can be an ultrasonic atomizer that can operate atfrequencies between 60 kHz and 120 kHz. The mounting assembly 11300 caninclude a support plate 11302 having coupling members 11305 attachedthereto. The support plate can include a bore 11306 that can receive theatomizer 11304, and an opening 11308 that can receive a retainingelement 11310 that can have the needle emitters 9002 coupled thereto.The bore 11306 can have a slot 11312 extending therefrom. In some cases,ultrasonic atomizers that operate at 60 kHz can have the same design asthose that operate at 120 kHz. Therefore, the mounting assembly 11300can accommodate ultrasonic atomizers that can operate at eitherfrequency.

FIG. 105 shows an exploded view of a portion of the mounting assembly11300 with the retaining element 11310 and the needle emitters 9002. Asshown in FIG. 105, the needle emitters 9002 can be inserted into bores11310a of the retaining element 11310, and the retaining element can beinserted into the opening 11308. The design of the retaining element11310 as a sliding insert can enable independent Z positioning, orvertical positioning, of the needle emitters 9002 relative to the sprayhead emitter. This can accommodate varied atomizer heights, in the range15-31 mm from a tip of the emitter to a base of a well of a filterplate, while maintaining a consistent needle emitter height.

FIG. 106 shows an example of a mounting assembly 11500 that canreleasably retain the needle emitters 9002 and an ultrasonic atomizer11504. The mounting assembly 11500 can generally be similar to mountingassembly 11300, but can be designed to function with an ultrasonicatomizer 11504 that can operate at 180 kHz. The mounting assembly 11500can include a support plate 11502 having coupling members 11505 attachedthereto. The support plate can include a bore (not shown) that canreceive the atomizer 11504, and an opening 11508 that can receive aretaining element 11510 that can have the needle emitters 9002 coupledthereto. The bore can have a slot 11512 extending therefrom.

In the illustrated embodiment, a collar 11514 can be attached to theatomizer 11504 and the collar slots in the same head holder at the sameheight and concentrically to the other two heads.

FIG. 107 shows an example of a mounting assembly 11600 that canreleasably retain the needle emitters 9002 and a nebulizer 11604. Themounting assembly 11600 can generally be similar to mounting assembly11300, and can include a support plate 11602 having coupling members11505 attached thereto. The nebulizer 11604 can be coupled to thesupport plate 11602 via a bracket 11606. The support plate 11602 caninclude an opening 11608 that can receive a retaining element 11610 thatcan have the needle emitters 9002 coupled thereto.

Similar design templates to those shown in FIGS. 78-81 can be used toaccommodate other emitter types.

The designs of the mounting assemblies 11300, 11500, and 11600 enablesthe precision rig system to perform as a platform for optimization ofthe delivery process. For example, 4 different spray heads can beaccommodated.

Automation and Software Design

The precision rig system 9000, and/or the positive pressure system 9300can include software program and user interface that can enableautomation of the delivery process steps. For example, the software canbe designed to control the translational stage 9010 and valves 9011. Asanother example, the software program can be configured to controlmovement of the mounting array 9302. A user interface can enable thecritical parameters to be entered. A sequence of functional units can beselected by the user.

A system was designed which had the capability of ensuring repeatabilityof experiments but can also provide the flexibility to adjustexperimental specific parameters. Such parameters can include the numberof wells one wants to address or the volume which is dispensed into awell. In order to achieve this synchronization, a traverse mechanism(e.g., the actuator 9319) is included, which can be software controlledto position its carriage within a 200 mm location for a required periodof time. The carriage includes 6 positions and each of these positionsare coupled to an individually controlled high precision fluidescapement chamber. It is possible to move each position under aselection of dispensing locations (e.g., Payload, Culture Medium andStop Solution stations). A device specific software platform can beincluded. The software platform can include: 1) a graphical userinterface which enables the user to design an experiment; and 2) localcontrol via a controller (e.g., programmable logic controller (PLC)),for example an Omron PLC. FIG. 129 shows a schematic 12000 of thesoftware platform design. The software can include a user friendlyexperiment creation section and a background sequence generator. Theuser interface can include an experiment canvas which allows the user tochange parameters in the experiment. The parameters, which can be variedby the user, include the location and number of wells to be addressed,the sequence of steps including vacuum and/or positive pressure,dispense of payload, stop solution and culture medium, and correspondingvolumes to be delivered. The user can also modify the actuator speed andthe incubation times (FIG. 130). FIG. 130 shows a portion of a graphicaluser interface (GUI) 12100 of the software platform. In the illustratedexample, the GUI includes an experiment canvas which allows the user tovary parameters. For example, the parameters which can be varied by theuser include the location and number of wells to be addressed, thesequence of steps including vacuum or positive pressure, dispensation ofpayload, stop solution, and/or culture medium, and the correspondingvolumes to be delivered. The background sequence generator outputs aprogram for the PLC, which provides local control of the mechanical andelectrical system. This software can be provided the operator (e.g., viaan integrated display) with the ability to test various permutation ofwells, media and time without having to manually calculate the dwelltime between wells for optimum experimental success. These inputs canprovide the end user with the capability of conducting multipleexperiments.

In some implementations, a system can address ninety six positionswithin a filter plate substrate. This system can be faster, havemultiple traverse mechanism and a user friendly HMI (human machineinterface). The HMI can provide the operator with additional automatedutilities such as automatic system purging or a touch screen drivencalibration sequence. This system can operate as a standalone solutionyet still maintained the link to the experimental designer for designingexperiment. More aspects of the experiment can be adjusted from well towell, which means the potential permutations greatly increases. Forexample fluid can be dispense to a number of wells and then the appliedpressure to the fluid delivery system for the next section of theexperiment can be automatically adjusted. This example system has thecapability of recording analytics from the experiments for furtheroffline analysis.

FIG. 64 is a process flow diagram illustrating an example processaccording to some aspects of the current subject matter. The exampleprocess can be implemented, e.g., by a controller of a delivery system.At 1610, user input can be received, e.g., by a controller. At 1620, thedelivery solution applicator can be operated to deliver atomizeddelivery solution to a cellular monolayer within a well. At 1630, thecellular monolayer can be incubated for a first period of time afterapplication of the delivery solution. At 1640, the stop solutionapplicator can be operated in response to expiration of the firstincubation period. The operation can be performed to deliver stopsolution to the cellular monolayer. At 1650, in response to applicationof the stop solution, the cellular monolayer can be incubated for asecond period of time.

In some implementation, the iteration of operation of the deliverysolution applicator, incubation for the first incubation period,operation of the stop solution applicator, and incubation for the secondincubation period for a predetermined number of iterations can beperformed.

One or more aspects or features of the subject matter described hereincan be realized in digital electronic circuitry, integrated circuitry,specially designed application specific integrated circuits (ASICs),field programmable gate arrays (FPGAs) computer hardware, firmware,software, and/or combinations thereof. These various aspects or featurescan include implementation in one or more computer programs that areexecutable and/or interpretable on a programmable system including atleast one programmable processor, which can be special or generalpurpose, coupled to receive data and instructions from, and to transmitdata and instructions to, a storage system, at least one input device,and at least one output device. The programmable system or computingsystem may include clients and servers. A client and server aregenerally remote from each other and typically interact through acommunication network. The relationship of client and server arises byvirtue of computer programs running on the respective computers andhaving a client-server relationship to each other.

These computer programs, which can also be referred to as programs,software, software applications, applications, components, or code,include machine instructions for a programmable processor, and can beimplemented in a high-level procedural language, an object-orientedprogramming language, a functional programming language, a logicalprogramming language, and/or in assembly/machine language. As usedherein, the term “machine-readable medium” refers to any computerprogram product, apparatus and/or device, such as for example magneticdiscs, optical disks, memory, and Programmable Logic Devices (PLDs),used to provide machine instructions and/or data to a programmableprocessor, including a machine-readable medium that receives machineinstructions as a machine-readable signal. The term “machine-readablesignal” refers to any signal used to provide machine instructions and/ordata to a programmable processor. The machine-readable medium can storesuch machine instructions non-transitorily, such as for example as woulda non-transient solid-state memory or a magnetic hard drive or anyequivalent storage medium. The machine-readable medium can alternativelyor additionally store such machine instructions in a transient manner,such as for example as would a processor cache or other random accessmemory associated with one or more physical processor cores.

To provide for interaction with a user, one or more aspects or featuresof the subject matter described herein can be implemented on a computerhaving a display device, such as for example a cathode ray tube (CRT) ora liquid crystal display (LCD) or a light emitting diode (LED) monitorfor displaying information to the user and a keyboard and a pointingdevice, such as for example a mouse or a trackball, by which the usermay provide input to the computer. Other kinds of devices can be used toprovide for interaction with a user as well. For example, feedbackprovided to the user can be any form of sensory feedback, such as forexample visual feedback, auditory feedback, or tactile feedback; andinput from the user may be received in any form, including, but notlimited to, acoustic, speech, or tactile input. Other possible inputdevices include, but are not limited to, touch screens or othertouch-sensitive devices such as single or multi-point resistive orcapacitive trackpads, voice recognition hardware and software, opticalscanners, optical pointers, digital image capture devices and associatedinterpretation software, and the like.

Enclosure for the Instrument

The precision rig system 9000, and/or the positive pressure system 9300can be retained within an enclosure to maintain stable ambientconditions. The enclosure can be customized base on parameters to affectthe design of the enclosure

Temperature and Control of the Base Plate

In some embodiments, a temperature control system can be implemented tocontrol the temperature of the base plate 9016. For example, cartridgeheaters can be installed at various locations on the base plate 9016 ofthe vacuum manifold assembly 9008. A thermodynamic analysis can beconducted to examine heat transfer from the cartridge heaters to thewell filter plate 9014. Temperature ranging experiments can be performedto investigate the effect of temperature on payload delivery.

Although a few variations have been described above, other modificationsare possible. For example, the filter plate can include any number ofwells, and need not be a 96-well filter plate. Additionally, the systemcan include any number of valves that can control vacuum pressure to anynumber of active wells. As another example, the system can include oneor more needle emitters, atomizers, and or nebulizers, each of which canbe independently mounted and controlled.

The current subject matter provides many technical advantages. Ingeneral, the current subject matter provides a delivery system thatenables greater consistency in the delivery process, and higherefficiency of delivery, while maintaining cell health.

The delivery system allows for vacuum pressure to be applied toindividual wells of a filter plate to remove a culture medium, therebycreating a monolayer of cells. By applying a vacuum pressure toindividual wells on a filter plate, greater precision, control of thevacuum pressure, and consistency of the vacuum pressure applied to eachwell, can be achieved.

The delivery system allows for dispensation of permeabilizing solutionin volumes on the order of microliters. Microliter dispensation volumesallow for greater control over cell exposure to the solution, which canincrease overall cell viability by reducing excessive exposure to thepermeabilizing solution. Moreover, the system can be automated whichminimizes error, and increases precision.

The system allows for control the temperature of the delivery solution,stop solution, and culture medium. Therefore, each solution can bemaintained at an optimum temperature to increase efficiency of payloaddelivery as well as cell viability. The temperature of the base platecan also be controlled. This allows for temperature optimization tomaximize efficiency of payload delivery.

The needle emitters and atomizer and/or nebulizer can be coupled to amounting assembly that can accommodate various atomizer/nebulizerheights, in the range 15-31 mm from a tip of the emitter to a base of awell of a filter plate, while maintaining a consistent needle emitterheight. This allows

The system can include hardware, one or more software programs, and auser interface, that can enable automation of the delivery processsteps. The software can be designed to control the translational stageand valves. A user interface can allow for critical parameters, such asto be entered. A sequence of functional units can be selected by theuser. This is beneficial because

The enclosure can function to maintain stable ambient conditions.

Additional Example Delivery System Aspects

Scaling the delivery process involved designing a system to enableoptimization, determining a method for formation of a cell monolayer ata larger scale, and optimizing atomisation to enable intracellulardelivery of mRNA to T-cells.

Optimization work to date has achieved >50% efficiency of mRNA deliveryto T-cells with >60% cell viability and cell recovery of up to 80%.

A system was designed and constructed to facilitate scaling of thedelivery process. The system is based around the commercially availableproduct, Amicon Stirred cell, pressure-based sample concentration unit(50 ml and 200 ml size; catalogue UFSC05001 and UFSC20001,respectively). FIG. 108 shows an example of a stirred cell system 9700configured to facilitate forming a monolayer of cells.

The stirred cell system 9700 can include a cap 9702, a body 9704, amembrane holder 9706, and a base 9708. A stir bar 9710 can be positionedwithin the body 9704. The cap 9702 can be configured to couple to thebody 9704 at a first end of the body 9704. A sealing element 9712 (e.g.,a gasket) can be positioned between the cap 9702 and the first end ofthe body 9704 to form a seal between cap 9702 and the first end of thebody 9704. The membrane holder 9706 can receive a membrane 9714, and canbe positioned adjacent to a second end of the body 9704 such that acoupling element 9716 of the membrane holder 9706 extends through anopening 9718 in the second end of the body 9704. The second end of thebody 9704 can be coupled to the base 9708, and a sealing element 9720(e.g., an o-ring) can be positioned therebetween to form a seal. Apressure inlet tubing assembly 9722 can be coupled to cap 9702 such thata pressurized gas can be delivered to the body a chamber formed by thecap 9702, the body 9704 and the base 9708. A filtrate tubing assembly9724 can be coupled to the coupling element 9716 of the membrane holder9706 such that fluid can be drained from the chamber upon application ofpressure.

In operation, a culture medium containing cells can be delivered to thechamber, and the cap 9702 can be coupled to the body 9704. The pressureinlet tubing assembly 9722 can be coupled to the cap 9702, and positivepressure can be applied to the chamber via the inlet tubing assembly9722. In some embodiments, the pressure can be in the range of 50-1000mbar, and the pressure can be applied for 10-60 seconds. Accordingly,upon application of positive pressure, the culture medium can be forcedthrough the membrane 9714 and out of the chamber via the couplingelement 9716 and the filtrate tubing assembly 9724. With the culturemedium evacuated, a monolayer of cells can remain on the membrane 9714.Accordingly, the stirred cell system 9700 can be used to form the cellmonolayer onto a filter membrane placed on the membrane holder of thestirred cell. With the monolayer formed, a nebulizer (e.g., the LB-100from Burgener Research), can be used to atomize the delivery solutiononto the cells sitting on the filter membrane.

The stirred cell system 9700, shown in FIG. 108, can have severallimitations. For example, in some cases, the stirred cell system 9700cannot filter culture mediums having cell suspensions above a certainconcentration (e.g., concentrations above 10×10⁶/mL when using whenusing a PCTE membrane). This may be, in part, due to the geometry of themembrane holder 9706. As shown in FIG. 109, the culture medium drainsthrough a single channel 9706 a of the membrane holder 9706. The channel9706 a can become clogged when filtering culture mediums having cellsuspensions above a certain concentration. As another example, an evendistribution of cells on the membrane 9714 was not achieved while usingthe membrane holder 9706.

To address these limitations with of membrane holder 9706, a membraneholder 9906, shown in FIG. 110, was constructed. In this embodiment, themembrane holder 9906 is made of PTFE, and it includes a plurality ofholes 9906 a drilled through its bottom surface in a pattern formingconcentric circles. When in use, a culture medium can drain through theholes 9906 a when pressure is applied within a chamber of a stirred cellsystem. The membrane holder 9906 was assessed using dynabeads(microbeads used to mimic the cells). FIG. 111 shows a membrane 9914that was used with the membrane holder 9906 during the dynabeadassessment. As shown in FIG. 111, the results indicate that dynabeadsaccumulated in the proximity of the holes 9906 a. Cells that weresubjected to the delivery process using this holder resulted in pooruptake.

In order prevent accumulation of cells near the holes, and to generate amore even distribution of cells on a membrane, a membrane holder caninclude holes similar to holes 9906 a, but can also include concentricchannels that can facilitate radial flow between the holes. FIG. 112shows another embodiment of a membrane holder 10006 that can be usedwith a stirred cell system (e.g. stirred cell system 9700). In theillustrated embodiment, the membrane holder 10006 includes holes 10006a, concentric channels 10006 b, and straight, or linear, channels 10006c. The holes 10006 a are formed along a pattern that includes a centralcircle, an outer circle, and linear spokes that extend from the centralcircle to the outer circle. The linear channels 10006 c extend radiallyoutward from a central point of the membrane holder 10006, and theconcentric channels 10006 b form concentric circles about the centralpoint of the membrane holder 10006. The holes 10006 a and channels 10006b, 10006 c can promote an even distribution of the cells over themembrane, and can promote rapid removal of the culture medium. In someembodiments, the membrane holder 9706, illustrated in FIGS. 82-83, canbe modified by drilling holes (e.g., the holes 10006 a), to form themembrane holder 10006.

The membrane holder 10006 was assessed using dynabeads. FIG. 113 shows amembrane 10014 that was used with the membrane holder 10006 during thedynabead assessment. As shown in FIG. 113, the results indicate that thedynabeads are more evenly distributed on the membrane 10014 that wasused with the membrane holder 10006 than they are on the membrane 9914that was used with the membrane holder 9906.

In some embodiments, a membrane holder can include holes formed withinconcentric channels. FIG. 114 shows an example of a membrane holder10106 that includes holes 10106 a, concentric channels 10106 b, andlinear channels 10106 c. In the illustrated example, the holes 10106 areformed within the concentric channels 10106 b and within the linearchannels 10106 c. This geometry can facilitate an even outflow of aculture medium such that a monolayer of cells can be formed on amembrane.

To enable atomisation of the delivery solution using a LB-100 sprayhead, R4 was built. FIGS. 115-118, and 128 show an example a solutiondelivery system, also referred to as R4 that can be used to deliver apermeabilizing solution to a monolayer of cells. The solution deliverysystem can include a nebulizer assembly 9801, shown in FIG. 115, and amounting system 9802, shown in FIG. 116.

The nebulizer assembly 9801 can include a nebulizer 9804 (e.g., theLB-100 spray head), a coupling element 9806 (e.g., an IDEX connection)configured to facilitate delivering air and liquid (e.g., thepermeabilizing solution) to the nebulizer 9804, a solution reservoir9810 (e.g., an Elveflow sample reservoir) configured to provide thepermeabilizing solution to the nebulizer 9804, and a pinch valve 9808configured to control delivery of the permeabilizing solution to thenebulizer 9804. The mounting system 9802 can include a valve andreservoir mount 9812, a spray head mount 9814, and a nebulizer retainingcollar 9816 to accommodate the nebulizer 9804. The nebulizer assembly9801 can be coupled to the mounting system 9082, as illustrated in FIGS.91-92, to be secured in place. The mounting system 9082 facilitatesaccurate alignment of the nebulizer 9804 with the target area.

Additional Example Approaches to Formation of a Cell Monolayer

Two approaches to creating a cell monolayer are described. In bothmethods, the stirred cell unit was assembled as demonstrated in FIG.108. A volume of 5-10 ml cell suspension containing 0.4-10×10⁶ cells/mlwas added to the stirred cell chamber and the lid of the chamber wasthen closed.

The monolayer can be created by applying vacuum pressure to the base ofthe stirred cell. In this method, −50 to −1000 mbar were applied to thechamber for 10-60 seconds or until the filter membrane appeared dry byeye.

Alternatively the monolayer was formed by applying a positive pressurethrough the tubing connection on the lid of the stirred cell. Pressurewas applied for a set time (10-60 s) or until the filter membraneappeared dry by eye. In some case a lower pressure was used todrive >90% of the culture medium through the filter and then thepressure was gently increased for 10-15 seconds at the end to achievecomplete removal of the culture medium. To form the monolayer andcompletely remove cell culture medium a pressure between 100-200 mbarwas applied.

The actual specific pressure and time varied depending on cellconcentration, membrane type, pore size and type of membrane holder. Forexample, with the original unmodified membrane holder, 50-100×10⁶T-cells were used to form a monolayer in the 63.5 mm stirred cell. Withthe PES (polyethylene sulfone) membrane, it was possible to remove themedia applying 150 mbar for 20-30 s in the case of 50×10⁶ cells while30-60 seconds and 200 mbar were necessary to remove the media on 60×10⁶cells. With the PCTE (polycarbonate track etched) membrane it took 60seconds at 250 mbar for the lower cell concentration. In the case of100×10⁶ cells after 2 min at 250 mbar the medium will still present onthe filter membrane. To remove this the pressure was raised to 1 bar for10 seconds and then decreased to normal levels (100 mbar). This was doneseveral times to aid media removal.

Positive pressure was tested with the unmodified filter holder (FIG.109) in the 63.5 mm and 44.5 mm stirred cell with ˜50×10{circumflex over( )}6 cells with the hydrophilic PCTE membrane with 2.0 μm pores. At nostage was all media removed with this setup (0-600 bar 0-6 mins)

With the modified filter holder (FIG. 113) in a 44.5 mm membrane amonolayer was achieved with as little as 100 mbar for 10 seconds in a 1μm PCTE hydrophobic membrane with 20×10⁶ cells but could be achievedwith less time at higher pressures (500 mbar 5 secs).

The type of filter used to generate the monolayer was investigated. Thefilters differed in material, hydrophobicity and pore size. Thematerials tested included PES and Polycarbonate track etched filters,PCTE, hydrophobic and hydrophilic membrane coating, sizes included 13,25 mm, 47 mm and 63 mm diameter and pore size ranged from 0.4 μm to 1.2μm diameter. Also tested were PETE (Polyester), Silver and Goldmembranes (See below Table).

TABLE Filter Type Pore size PES 0.8, 1.2, 3, 5.0 PCTE Hydrophobic 0.4,0.8, 1, 3 PCTE Hydrophilic 0.4, 0.8, 1, 2, 3 PETE 0.2, 1.0, 2.0

The filters were assessed for formation of an even monolayer, efficiencyin removal of the culture medium and recovery of the cells from thefilter membrane. Dynabeads were used as representative of cells toassess monolayer formation. In addition, expanded T-cells were used toassess monolayer formation, recovery and viability post monolayerformation.

The PES filters were investigated and it was found these filters enabledformation of an even monolayer and efficient removal of the culturemedium. However, recovery of cells from the filter membrane was low(50%). The PCTE, track-edge filters, resulted in improved cell recovery(50-90%) from the filter membrane (FIG. 121). However, the efficiency ofculture medium removal was slower compared to the PES filters (45seconds to remove medium from PCTE filters compared to 10 seconds withthe PES filter (at 150 mbar)). The best recovery that was achieved was˜87% with PCTE 0.4 Hydrophobic 20×10⁶100 mbar for 60 seconds (FIG. 122).

Atomization to Enable Intracellular Delivery of mRNA to T-Cells

Investigation of the LB-100 spray head demonstrated a target area of 60mm in diameter. Force analysis of the LB-100 was carried out (FIG. 120).Height, air pressure, volume delivered, number of hits, cell number andspray duration were adjusted to optimise delivery with the LB-100 sprayonto a larger target area (25-65 mm filter membrane).

To optimise LB-100 spray delivery, the height of the spray head to thetarget area was adjusted. Distance in the range of 30 mm to 160 mm fromspray head tip to the target area was investigated. The optimal range ofdistance was found to be within 60 to 100 mm from tip of the spray headto the target area. Uptake increased at the lower distance and decreasedas you moved further away.

To optimise LB-100 spray delivery, the air pressure was varied in therange of 1 to 6 bar, with 2.5 to 3.0 bar found to be the optimal rangefor intracellular delivery. GFP mRNA delivery was reduced at pressureslower than 2.5 bar and cell viability was reduced at pressures higherthan 3.0 bar. Increasing the air pressure did increase the effectivetarget area.

To optimise LB-100 spray delivery, the volume delivered was varied inthe range of 10 to 300 μl, with a volume between 80 to 100 μl found tobe optimal for intracellular delivery. 5-10 ml cell suspension of humanprimary T cells in the range of 0.4-10×10⁶/ml was added to the stirredcell unit (Merck Millipore; PCTE filter, 0.4 μm or 1 μm pore size).Positive pressure in the range of 100-150 mbar was applied for 20-50 sto form the cell monolayer. The cell monolayer was sprayed with 10-300μl of delivery solution containing 0.1 μg/μl of GFP mRNA and incubatedfor 2 min. Stop solution (1 ml) was added and incubated for 30 sfollowing this, culture medium (4 ml) was added to the filter membrane.Cells were incubated overnight at 37° C. and 5% CO₂ in a humidifiedincubator and assessed for GFP fluorescence by flow cytometry between17-24 hours later.

To increase delivery efficiency, an assessment of the optimal number ofhits comparing 1 and 2-hit strategies was performed. Data demonstratedan increase in delivery efficiency when cells received a second hit.5-10 ml cell suspension of human primary T cells in the range of0.4-10×10⁶/ml was added to the stirred cell unit (Merck Millipore; PCTEfilter, 0.4 μm or 1 μm pore size). Positive pressure in the range of100-150 mbar was applied for 20-50 s to form the cell monolayer. Thecell monolayer was sprayed with 80-100 μl of delivery solutioncontaining 0.1 μg/μl of GFP mRNA and incubated for 2 min. Stop solution(1 ml) was added and incubated for 30 s following this, culture medium(4 ml) was added to the filter membrane. For the 2-hit strategy thecells were incubated for 2 hours before the spray process was repeated(as described above). Cells were incubated overnight at 37° C. and 5%CO₂ in a humidified incubator and assessed for GFP fluorescence by flowcytometry between 17-24 hours later. The double hit process resulted inincreased delivery efficiency (FIG. 123).

FIG. 132 shows an exemplary embodiment of a Midi system 12300. As shownin the illustrated example, the Midi system 12300 includes a stirredcell system 11900 (e.g., a 63 mm stirred cell system) with membraneholder 10006 (e.g., a 44 mm membrane holder). An enclosing film 12303 isadhered to an opening of the stirred cell unit. A collar of a spray headholder 9814 has been inserted into stirred cell system 11900 through a‘slit’ in the enclosing film 12303. In the illustrated example, a LB-100atomizer is retained within the collar of the spray head holder 9814,and it is positioned such that a tip of the spray head is 82 mm from anupper surface surface of the membrane holder 10006.

FIG. 133 is a plot showing data characterizing efficiency (GFP uptake)and viability corresponding to tests performed with the Midi system12300. The data demonstrates an average delivery efficiency of59.63%±1.2 and average viability data of 74.6%±5.3 across 3 technicalrepeats.

To increase delivery efficiency, the number of cells seeded on thefilter membrane was investigated. Cell density of 13×10³ cells/mm² wereseeded in the stirred cell unit (Merck Millipore; PCTE filter, 0.4 μm or1 μm pore size). Positive pressure in the range of 100-200 mbar wasapplied for 10-50 s to form the cell monolayer. The cell monolayer wassprayed with 80-100 μl of delivery solution containing 0.1 μg/μl of GFPmRNA and incubated for 2 min Stop solution (1 ml) was added andincubated for 30 s, followed by addition of culture medium (4 ml) to thefilter membrane. Cells were incubated overnight at 37° C. and 5% CO₂ ina humidified incubator and assessed for GFP fluorescence by flowcytometry between 17-24 hours later.

The duration of the spray was investigated. This was achieved byadjusting the opening time of the valves which control the flow of airand payload to the atomiser. Spray duration between 280-700 ms weretested.

FIG. 134 shows an exemplary embodiment of a delivery system configuredto facilitate generating a monolayer of cells and delivering a payloadto cells.

In some embodiments, to facilitate and to enhance the exposure of cellsto permeabilizing solution a filter membrane can be vibrated before,after, and/or during, delivery of the permeabilizing solution. To assistin the formation of a monolayer of cells on a filter membrane, themembrane can be vibrated before, after, and/or during, formation of themonolayer.

The vibration may be brought about by an eccentric rotating mass (ERM)system or a linear resonant actuator (LRA) system. For example, in apreferred embodiment, 1, 2 or 3 actuators (LRA) corresponding to the X,Yand Z axis can be attached to the membrane or a corresponding membraneholder (e.g., membrane holders 10006, 10106) such that the membranevibrates when the actuators are activated. An advantage of the LRAs isthat each axis of vibration can be driven independently. Accordingly,controllable vibration patterns may be developed on the membrane.Additionally, identification of mechanical resonance points due tophysical characteristics of the membrane can improve a degree of controlthat can be exhibited over the membrane. In some embodiments, a 3 axesaccelerometer device can be mechanically coupled to the membrane and/ormembrane holder to provide data characterizing motion and/or excursionof the membrane and/or membrane holder. Data from the accelerometer canbe used within a feedback control system to control actuation of theLRAs. For example, the accelerometer can be used to monitor vibrationsof the membrane and/or membrane holder. In some embodiments, data fromthe accelerometer can be used as a control feedback signal to adjustvibrations generated by the LRAs. For example, data from theaccelerometer can be used to generate an error signal between a desiredvibrational pattern and an achieved vibrational pattern. As an example,driving vibrational frequencies can be determined based on a stiffnessof the membrane and/or sizes of cells on the membrane. An examplevibrational pattern can brought about with sinusoidal signals at 3000 Hzon the x and y axes and no signal on the z axis. The excursions can be 1mm peak to peak and the x and y driving waveforms can be coherent withno phase difference between them. Many other patterns are possibleincluding ones that lead to swirling and/or shaking in the x, y, and/orz axes.

The current subject matter can include a non-viral, vector-free methodthat achieves intracellular delivery through gentle reversiblepermeabilization. The current subject matter can include 1) apermeabilising solution that contains a low dose of ethanol as thepermeabilising agent and 2) a means of applying the delivery solution tothe target cells in a dropletised form. The technology provides tightcontrol over the volume, time and pressure at which the permeabilisingsolution is applied to the cells and this enables high levels ofdelivery efficiency as well as cell viability to be attained.

The steps of the process can include: a monolayer of target cells isgenerated; supernatant is removed from the cells; the cargo is mixedwith the delivery solution and applied to the cells in a dropletisedform and incubated for 2 min; during this period, the ethanolpermeabilises the cell membrane and the cargo diffuses into the cell;cargo enters directly into the cytoplasm in an endocytosis-independentmanner; a ‘stop’ solution is then applied to the cells and incubated for30 sec—this acts to dilute the permeabilising delivery solution andallows the cell membrane to begin to reseal; culture medium is thenadded to complete the process.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the invention.

1. A method of delivering a payload across a plasma membrane of a non-adherent cell, comprising, providing a population of non-adherent cells; and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution including the payload and an alcohol at greater than 5 percent (v/v) concentration.
 2. The method of claim 1, wherein said alcohol comprises ethanol.
 3. The method of claim 2, wherein said aqueous solution comprises greater than 10% ethanol.
 4. The method of claim 2, wherein said aqueous solution comprises between 20-30% ethanol.
 5. The method of claim 1, wherein said aqueous solution comprises 27% ethanol.
 6. The method of claim 1, wherein said aqueous solution comprises between 12.5-500 mM KCl.
 7. The method of claim 1, wherein said aqueous solution comprises 106 mM KCl.
 8. The method of claim 1, wherein said non-adherent cell comprises a peripheral blood mononuclear cell.
 9. The method of claim 1, wherein said non-adherent cell comprises an immune cell.
 10. The method of claim 1, wherein said non-adherent cell comprises a T lymphocyte.
 11. The method claim 9, wherein said immune cell is activated with a ligand of CD3, CD28, or a combination thereof.
 12. The method of claim 1, wherein said population of non-adherent cells comprises a monolayer.
 13. The method of claim 1, wherein said monolayer is contacted with a spray of said aqueous solution.
 14. The method of claim 1, wherein said method delivers said payload into the cytoplasm of said cell and wherein said population of cells comprises a greater per cent viability compared to delivery of said payload by electroporation.
 15. The method of claim 1, wherein said payload comprises a messenger ribonucleic acid (mRNA).
 16. The method of claim 5, wherein said mRNA encodes a gene-editing composition.
 17. The method of claim 16, wherein said gene editing composition reduces the expression of PD-1.
 18. The method of claim 13, wherein said monolayer resides on a membrane filter.
 19. The method of claim 13, wherein said membrane filter is vibrated following contact with said spray.
 20. The method of claim 15, wherein said mRNA encodes a chimeric antigen receptor.
 21. A system comprising: a housing configured to receive a plate comprising a well; a differential pressure applicator configured to apply a differential pressure to the well; a delivery solution applicator configured to deliver atomized delivery solution to the well; a stop solution applicator configured to deliver a stop solution to the well; and a culture medium applicator configured to deliver a culture medium to the well.
 22. The system of claim 21, further comprising: an addressable well assembly configured to: align the differential pressure applicator adjacent the well for applying the differential pressure to the well; align the delivery solution applicator adjacent the well for delivering the atomized delivery solution to the well; align the stop solution applicator adjacent the well to deliver the stop solution to the well; and/or align the culture medium applicator adjacent the well to deliver the culture medium to the well.
 23. The system of claim 22, wherein the addressable well assembly includes a movable base-plate configured to receive the plate comprising the well and move the plate in at least one dimension.
 24. The system of claim 22, wherein the addressable well assembly includes a mounting assembly configured to couple to the delivery solution applicator, the stop solution applicator and the culture medium applicator.
 25. The system of claim 21, wherein the delivery solution applicator includes a nebulizer.
 26. The system of claim 21, wherein the delivery solution applicator is configured to deliver 10-300 micro liters of the delivery solution per actuation.
 27. The system of claim 21, further comprising a temperature control system configured to control a temperature of the delivery solution and/or of the plate comprising the well.
 28. The system of claim 21, further comprising an enclosure configured to control an environment of the plate comprising the well.
 29. The system of claim 21, wherein the differential pressure applicator comprises a nozzle assembly configured to form a seal with an opening of the well and to deliver a vapor to the well to increase or decrease pressure within the well, thereby driving a liquid portion of the culture medium from the well such that a layer of cells remains within the well.
 30. The system of claim 21, wherein the stop solution applicator comprises a needle emitter configured to couple to a stop solution reservoir.
 31. The system of claim 21, wherein the culture medium applicator comprises a needle emitter configured to couple to a culture medium reservoir.
 32. The system of claim 21, further comprising a controller configured to: receive user input; operate the delivery solution applicator to deliver the atomized delivery solution to a cellular monolayer within the well; incubate, for a first incubation period, the cellular monolayer after application of the delivery solution; operate, in response to expiration of the first incubation period, the stop solution applicator to deliver the stop solution to the cellular monolayer; and incubate, for a second incubation period and in response to application of the stop solution, the cellular monolayer.
 33. The system of claim 22, wherein the controller is further configured to: iterate operation of the delivery solution applicator, incubation for the first incubation period, operation of the stop solution applicator, and incubation for the second incubation period for a predetermined number of iterations.
 34. The system of claim 21, further comprising a controller configured to: operate the positive pressure system to remove supernatant from the well to create a cellular monolayer within the well.
 35. The system of claim 21, wherein the delivery solution applicator includes a spray head and a collar encircling a distal end of the spray head, wherein the collar is configured to prevent contamination between wells in a multi-well plate, wherein the collar is configured to provide a gap between the plate and the collar.
 36. The system of claim 35, wherein the delivery solution applicator includes a spray head and a film encircling a distal end of the spray head.
 37. The system of claim 21, further comprising a vibration system coupled to a membrane holder and configured to vibrate a membrane.
 38. The system of claim 21, further comprising: the plate, wherein the well is configured to contain a population of non-adherent cells.
 39. The system of claim 21, wherein the delivery solution includes an isotonic aqueous solution, the aqueous solution including the payload and an alcohol at greater than 5 percent (v/v) concentration.
 40. The system of claim 38, wherein said alcohol comprises ethanol.
 41. The system of claim 39, wherein said aqueous solution comprises greater than 10% ethanol.
 42. The system of claim 39, wherein said aqueous solution comprises between 20-30% ethanol.
 43. The system of claim 38, wherein said aqueous solution comprises 27% ethanol.
 44. The system of claim 38, wherein said aqueous solution comprises between 12.5-500 mM KCl.
 45. The system of claim 38, wherein said aqueous solution comprises 106 mM KCl.
 46. The system of claim 37, wherein said non-adherent cell comprises a peripheral blood mononuclear cell.
 47. The system of claim 37, wherein said non-adherent cell comprises an immune cell.
 48. The system of claim 37, wherein said non-adherent cell comprises a T lymphocyte.
 49. The system of claim 38, wherein said payload comprises a messenger ribonucleic acid (mRNA).
 50. The system of claim 49, wherein said mRNA encodes a gene-editing composition.
 51. The system of claim 50, wherein said gene editing composition reduces the expression of PD-1.
 52. The system of claim 49, wherein said mRNA encodes a chimeric antigen receptor.
 53. The system of claim 21 for use to deliver a cargo compound or composition to a mammalian cell.
 54. The system of claim 37, wherein said population of non-adherent cells comprises a monolayer.
 55. A composition comprising an isotonic aqueous solution, the aqueous solution comprising KCl at a concentration of 10-500 mM and and ethanol at greater than 5 percent (v/v) concentration for use to deliver a cargo compound or composition to a mammalian cell.
 56. The composition of claim 55, wherein said KCl concentration is 106 mM and wherein said alcohol concentration is 27%.
 57. (canceled) 